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Full text of "Actin-based motility : functional analysis of pathogenic bacteria and host cell proteins"

ACTIN-BASED MOTILITY: FUNCTIONAL ANALYSIS OF PATHOGENIC 
BACTERIA AND HOST CELL PROTEINS 



By 
WILLIAM L. ZEILE 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

1997 



The work and the contribution to science that this dissertation represents would not 
exist if it were not for my family. This is dedicated to Patricia Zeile, my wife and our four 
wonderful children; Kathleen Ruth Zeile, James Walter Zeile, Rachel Euseba Zeile, and 
Joshua William Zeile. Their joy for living, patience, and love has been an unending source 
of support and encouragement. 



ACKNOWLEDGMENTS 

It is a true joy to learn at the hands of great leaders in science. I would like to give 
very special thanks to my co-mentors, Dr. Frederick Southwick and Dr. Daniel Purich for 
allowing me the opportunity to work in their laboratories and study under their direction. I 
would also like to thank my other committee members: Dr. Gudrun Bennett, Dr. Brian 
Cain, and Dr. Charles Allen, for their interest and creative suggestions. I would like to 
especially thank Dr. Fan Kang and Dr. Ron Laine for their help and friendship and for 
being daily reminders for what is best in experimental science. 

Finally, I would like to thank, Patty, my wife of 19 years. We have been through 
an incredible life journey during those years. At each stage, from the Peace Corps, to dairy 
farming, to graduate school, Patty has been a true partner. With her clever mind, good 
sense of humor, love, and encouragement, Patty, who has always recognized what made a 
fulfilling and happy life, has shared equally with me in reaching our career and life goals. I 
would like to thank my children, Katie, James, Rachel, and Josh, for all their love, help, 
and encouragement. 



in 



i 



TABLE OF CONTENTS 

page 

ACKNOWLEDGMENTS iii 

LIST OF FIGURES vi 

ABSTRACT viii 

CHAPTERS 

1 INTRODUCTION: ACTIN-BASED MOTILITY OF BACTERIAL 

PATHOGENS 1 

Specific Aims and Findings 1 

Background and Significance 3 

Actin and the Actin-Based Cytoskeleton 4 

Actin Binding Proteins Important in Actin-Based Motility 6 

Life Cycle and Actin-Based Motility of Intracellular Pathogens 8 

A model for Actin-Based Motility of Bacterial Pathogens 13 

Summary j5 

2 RECOGNITION OF TWO CLASSES OF OLIGOPROLINE SEQUENCES IN 

PROFILIN-MEDL\TED ACCELERATION OF ACTIN-BASED SHIGELLA 
MOTILITY 16 

Introduction 15 

Materials and Methods 21 

Materials 21 

Tissue Culture Methods and Infection Procedures 21 

Microscopy and Microinjection 22 

Results 27 

Characteristics of Shigella Movement and Actin Rocket-Tail Formation in 

PtK2 Cells 27 

Fluorescence Staining of Actin and Alpha- Actinin in Shigella Rocket-Tails 27 

Arrest of Shigella Intracellular Movement by the Second Oligo-Proline 

Repeat Analogue in Listeria ActA Protein 29 

Effect of Microinjecting a Binary Solution of Profilin and ActA Analogue on 

Shigella Intracellular Movement 29 

Effects of Microinjection of a VASP Oligoproline Analogue Alone and in 

Combination with Profilin on Shigella Intracellular Motility 32 

Discussion 35 

3 VINCULIN PROTEOLYSIS UNMASKS AN ACTA HOMOLOGUE FOR 

ACTIN-BASED 5///G£:LM MOTILITY 43 



IV 



Introduction 43 

Materials and Methods 48 

Materials 48 

Antibodies 48 

Purification of Recombinant IcsA 49 

Affinity Chromatography 50 

Solid Phase Overlay Assay 50 

Microinjection Experiments 51 

Vinculin Proteolysis after Shigella Infection of PtK2 Cells 51 

Indirect Immunofluorescence Microscopy 52 

Results 54 

Anti-ActA-peptide Immunofluorescence Microscopy localizes a Cross- 
Reactive Protein at the Back of Moving Bacteria 54 

IcsA Affinity Chromatography and Solid Phase Binding Assays Fail to 

Isolate or Identify an ActA Mammalian Homologue 55 

Identification of a Cleaved Form of Vinculin, p90 as the ActA Mammalian 

Homologue 55 

Vinculin's Head Domain Localizes to the Surface of Intracellular Shigella 59 

The Vinculin Head-Fragment is Generated after Shigella Infection 61 

Discussion 63 

4 PRELIMINARY STUDIES OF HOST CELL VINCULIN AND SHIGELLA 

ICSA BINDING INTERACTIONS 69 

Introduction 69 

Materials and Methods 71 

Cloning of Glutathione S-Transferase Fusion Protein cDNA Expression 

Constructs 71 

Recombinant Protein Purification 72 

In vitro Solution Phase Binding Assay: Bead Binding Assay 73 

Results 74 

Construction and Expression of GST-IcsA Fusion Proteins 74 

Solution Phase Binding Assay by Co-Preciptiation with Glutathione 

Sepharose Beads 81 

Binding of Vinculin to Shigella IcsA Requires the Glycine-Rich Region of 

IcsA and Cleavage of Vinculin to the p90 Form 81 

Discussion 85 



5 SUMMARY AND FUTURE DIRECTIONS 89 

In vitro Expression Cloning to Isolate a Specific Protease 90 

Biopanning a Phage Display Library 92 

Biopanning a Phage Display Library Based on a Biased Oligonucleotide 

Sequence 93 

Another Model System: Vaccinia Virus 93 

The Actin-Based Motility Complex 95 

LIST OF REFERENCES 99 

BIOGRAPHICAL SKETCH 106 



LIST OF FIGURES 

Fi gure page 

1.1. The two steps of actin filament assembly 5 

1 .2. Intracellular Life-cycle of Listeria 9 

1.3. Schematic model of ActAA^ASP/profilin interactions leading to the generation of 
ATP-actin monomers 14 

2. 1 . Phase images of Shigella rocket tails 20 

2.2. Comparison of bodipy-phallacidin staining of Shigella and Listeria actin filament 
rocket tails 24 

2.3. Anti-alpha actinin immunofluorescence and bodipy-phallacidin fluorescence images 
of Shigella rocket tail 26 

2.4. Shigella movement and actin rocket tail formation in PtK2 host cells before and after 
microinjection of the synthetic ActA peptide 28 

2.5. Effects of microinjection of the ActA analogue on Shigella motility 30 

2.6 A. Time-lapse phase micrograph of Shigella motility in a PtK2 cell before and after 
microinjection of an ActA analogue/profilin binary solution 31 

2.6 B. Velocities of two bacteria in a PtK2 cell before and after the microinjection of an 
ActA analogue/profilin binary solution 32 

2.7. The effect of microinjection of VASP peptide analogue on Shigella motility in a 
PtK2 cell 33 

2.8. Microinjection of poly-L-proline, profilin and mixtures of VASP analogue and 
profilin and mixtures of poly-L-proline and profilin on Shigella motility 35 

2.9. Working model showing the primary components likely to be involved in the actin- 
based locomotory unit of Shigella 42 

3.1. Characteristics of anti-ActA antibody 47 

3.2. Structural organization of human vinculin 53 

3.3. Immunofluorescence microscopy of ^/z/geZ/a-infected PtK2 cells using 
anti-vinculin antibody 58 

3.4. Effect of Vine- 1 peptide on Shigella speed 60 



VI 



3.5. Shigella infection induces the production of the 90 kDa vinculin head-fragment ... 62 

3.6. A working model for vincuHn proteolysis and assembly of the Shigella actin- 

based motility complex 58 

4. 1 . Schematic representation of Shigella IcsA 74 

4.2. Schematic of GST-IcsA fusion proteins used in this study and expression in 

E. coli 77 

4.3. Purification of GST-IcsA fusion proteins 80 

4.4. Vinculin p90 binds to full length IcsA and to glycine-rich domain of IcsA 83 

5.1. Schematic of Actin-Based Motility Complexes of Listeria and Shigella actin 

motors 98 



vu 



Abstract of Dissertation Presented to the Graduate School 

of the University of Florida in Partial Fulfillment of the 

Requirements for the Degree of Doctor of Philosophy 

ACTIN-BASED MOTILITY: FUNCTIONAL ANALYSIS OF PATHOGENIC 
BACTERIA AND HOST CELL PROTEINS 

By 

William L. Zeile 
December 1997 



Chairman: Frederick Southwick, M.D. 

Major Department: Biochemistry and Molecular Biology 

The molecular architecture of the eukaryotic cell is, in part, determined by the actin- 

based cytoskeleton. Understanding the dynamics of this structure is key to understanding 

many cellular processes and may lead to discoveries for the treatment of diseases that are 

caused by a breakdown of the control and regulation of actin polymerization. The 

intracellular bacterial pathogens. Listeria monocytogenes and Shigella flexneri, during 

infection of host cells remodel the actin microfilaments; this remodeling supports bacterial 

motility and spread from cell to cell. We have used the infection of host cells by these 

organisms as a model system for studying the molecular mechanism of actin-based 

motility. In so doing we have addressed three hypothesis: 1) Listeria and Shigella share a 

similar mechanism of actin assembly and parasitism of the host by these organisms requires 

host cell proteins in addition to host cell actin. 2) For actin-based motility of Shigella, an 

additional host cell adapter protein is essential. 3) This host cell adapter protein must be 

activated by proteolysis during infection for productive assembly of the Shigella actin 

motor. Microinjection of infected mammalian tissue culture cells using specific competitor 

peptides designed to mimic the binding domains of the Listeria ActA surface protein and the 

host cell protein, vasodilator-stimulated phosphoprotein (VASP), uncoupled the actin 



vm 



motors of Listeria and Shigella. These experiments demonstrated a shared mechanism of 
actin assembly by these bacteria. The surface protein IcsA of Shigella required for actin- 
based motiUty has no homology to the Listeria ActA protein yet their mechanisms are 
similar. We determined that a host cell adapter protein containing ActA-like sequences was 
required for Shigella motility, and by immunological methods we discovered the 
cytoskeletal protein vinculin to be this protein. For productive assembly of the actin motor 
of Shigella, vinculin is proteolyzed to an active state in which the ActA-like sequence is 
unmasked for binding VASP. Activation of vinculin by a specific protease is likely the 
result of an apoptotic cascade initiated by the bacterium upon infection. Assembly of a 
competent actin motor may involve an ordered mechanism; understanding this should teach 
us how the cell regulates and assembles higher order molecular structures. 



IX 



CHAPTER 1 
INTRODUCTION: ACTIN-BASED MOTILITY OF BACTERIAL PATHOGENS 

Specific Aims and Findings 

We would like to understand how during infection of host cells, the bacterial 
pathogens Listeria monocytogenes and Shigella flexneri take over the actin cytoskeleton, 
and direct the polymerization of actin. By doing so, they are able to move within the 
cytoplasm and spread from cell to cell. The ability to spread from cell to cell explains many 
of the unique clinical manifestations of listeriosis and shigellosis and this step is absolutely 
required to cause disease. Both of these intracellular pathogens spread from cell to cell by 
inducing a host cell protein, actin, to polymerize into filaments or "rocket tails", which 
propel these organisms through the cytoplasm to the peripheral membrane. Here, the rocket 
tails drive the bacteria into filopods (membrane protrusions) which can be ingested by 
adjacent cells, allowing the organisms to pass from cell to cell without being exposed to the 
humoral immune system or extracellular antibiotics. This strategy explains, in particular. 
Listeria's predisposition to infect individuals with impaired cell-mediated immunity 
including pregnant women, neonates, organ transplant patients, and persons with AIDS. 
This same strategy also explains the rapid colonization and resulting devastation of the 
intestinal epithelial cell layer by Shigella during infection. 

The first aim of this research was to address the hypothesis that Listeria and 
Shigella share a common component or components in the mechanism of induced actin 
polymerization or rocket tail formation. Recent studies in our laboratory and others had 
shown that Listeria's ability to attract VASP from its normal host cell binding sites is the 
first step in usurping host cell actin assembly (Chakraborty et al, 1995). The oligoproline 
sequence repeat FEFPPPPTDE of Listeria's Act A surface protein, hereafter known as an 



1 



ABM-1 site (for Actin Based Motility l)(Purich and Southwick, 1997), serves as the 
critical VASP-binding site. If this sequence was important for Listeria, might a similar 
docking site be found on a protein necessary for Shigella motility as well? Using synthetic 
peptides specific to the above sequence, control peptides, and purified cytoskeletal 
components, microinjection experiments were carried out which demonstrated a shared 
mechanism, based on the ABM-1 site, for actin-based motility by Listeria and Shigella. 

The second aim of this research was to investigate the hypothesis that the 
mammahan host cell provides a component containing an ABM-1 site that Shigella usurps 
during infection. We would identify this ABM-1 containing component and try to 
understand the mechanism by which this host cell protein could become available for actin 
assembly during infection by Shigella. The cytoskeletal protein, vinculin, was identified as 
this component essential for 5/irge/Za motility. It had the critical ABM-1 site and the 
mechanism by which it becomes available to the bacterium during infection was found to be 
cleavage by limited proteolysis. This cleavage results in the unmasking of the critical 
ABM-1 site necessary for VASP binding. 

The hypothesis that vinculin, when activated by proteolysis, directly binds to the 
surface protein of Shigella, IcsA, during assembly of the actin-based motor was addressed 
as the third and final aim of this research. In vitro binding studies and chemical 
crosslinking demonstrated that host cell vinculin binds IcsA with high affinity and that this 
protein-protein interaction takes place within the amino terminal glycine-rich region of 
IcsA. These studies reinforced our earlier findings and demonstrated that vinculin must be 
activated by limited proteolysis for productive binding to Shigella IcsA. 

This research has allowed us to dissect the cascade of protein binding interactions 
required to generate Shigella 's and Listeria's actin-based motor, adding to the knowledge 
of how virulent pathogenic bacteria invade and spread cell to cell. This promises to advance 
preventive or remedial measures for diseases caused by these organisms. 



Background and Sig nificance, 

Bacterial infections of humans in which the pathogenic organism invades and 
multipHes intracellularly are a leading cause of disease and death in the world population. 
The bacteria of these infections not only colonize the external linings of the gut and 
airways, but direcdy invade the epithehal cells that line these passages causing 
inflammation and abscesses. Depending on the pathogen these organisms can spread cell 
to cell, exit the host cells, migrate through the extracellular matrix, and enter the circulation. 
Some members of this special class of intracellular bacteria are Mycobacterium leprae, the 
cause of leprosy with 12 million individuals affected worldwide; Mycobacterium 
tuberculosis, the cause of tuberculosis with 10 million new cases per year; and Chlamydiae 
trachomatis, a leading cause of blindness. Other members of this class of parasites cause 
Legionnaire's disease, salmonellosis, psittacosis, tularemia, brucellosis, listeriosis, and 
shigellosis (Cossart and Mengaud, 1995). 

Of these, the last two are responsible for increased morbidity and mortality in the 
world. Shigellosis is estimated to cause over 100 million cases of dysentery and over 
600,000 deaths a year (Gyles, 1993). Gram negative bacteria of the Shigella genus are 
found in the intestinal tracts of man and animals and are readily spread by fecal contact. 
Shigellosis is a deadly dysentery of children and the elderly, especially in the less 
developed countries of the world, where death from the infection comes mainly from 
dehydration. 

Listeriosis to a healthy individual poses little threat, but to pregnant women, the 
elderly, and the immuno-compromised (such as AIDS patients), the disease is serious and 
can lead to bacterial meningitis and death. Listeria monocytogenes, a gram positive rod, 
the only member of the Listeria genus to infect humans, is a common organism found in 
soil, water, plants, and the intestinal tracts of many animals. This organism as a 
contaminant of food is of increasing concern to public health officials as a Listeria infection 



has a 23% mortality as opposed to infections from other food-borne organisms which are 
rarely fatal (Southwick andPurich, 1996). 

Understanding the mechanisms by which intracellular pathogens invade and infect 
man will lead to methods for prevention and cure of the diseases caused by these parasites. 
To the cell biologist studying the cytoskeleton, the bacteria Listeria monocytogenes and 
Shigella flexneri open a unique window into the mechanisms of cytoskeletal remodeling 
that are operating in many eukaryotic cell types. These intracellular bacteria are members of 
a distinct subclass of invasive bacteria that upon entry into a host cell are able 1) to usurp 
the host cell cytoskeleton, 2) to support motility through the cytoplasm and, 3) to invade 
neighboring cells. This unique adaptation of remaining intracellular bacteria throughout 
most of their life cycle allows these bacteria to multiply and spread undetected by the host 
humoral immune system. What is required of the bacteria is the ability to stimulate actin 
polymerization in an ordered and directed manner to allow the polymers of actin, or 
microfilaments, to form a solid support in the cytoplasm by which the bacteria can be 
propelled forward. The obvious question is how do the bacteria accomplish this? Will 
understanding actin-based motility of bacteria add to our understanding of actin-based 
cytoskeletal remodeling in highly motile cells such as macrophages and neutrophils and in 
less motile, but equally dynamic, cells such as fibroblasts and epithelial cells? Much is 
known of actin dynamics in vitro and many actin associated proteins have been identified 
and been given functions in the cell. Very little is known about the mechanism of actin 
assembly in the cell, how that process is regulated, and what part all the various actin 
associated proteins play in assembling and maintaining the microfilament cytoskeleton. It 
is for these reasons that we study the actin-based motor of Listeria and Shigella as a model 
system. 
Actin and the Actin-Based Cytoskeleton 

The cytoskeleton of eukaryotic cells is made up of three main structural elements: 
the microtubules, the intermediate filaments, and the microfilaments. Whereas the 



microtubules form a framework for molecular transport in the non-mitotic cell and a 
framework for chromosomal transport in the mitotic cell, and the intermediate filaments 
form a stable, less dynamic scaffold, the microfilaments support a highly dynamic 
architecture for cellular remodeling and movement. The naicrofilaments are polymers of the 
43 kD protein, actin, and are found in muscle as well as non-muscle cells. Actin exists as a 
globular monomer (G-actin) or in filaments (F-actin) in which the monomers are 
assembled in a head to tail arrangement. Although there are different isoforms in different 
cell types, monomeric actin has a primary sequence of 375 residues and in tertiary structure 
is made up of a large and small domain separated by a cleft which serves as a nucleotide 
binding site and cation binding site (Kabsch et al., 1990). Nucleotide binding to actin is 
either in the form of adenosine triphosphate (ATP) or adenosine diphosphate (ADP). The 
myosin heavy chain subfragment S 1 binds to the small domain of actin and in muscle this 
binding supplies the necessary contact in muscle contraction. 



IMuc:l«:art i on 

ATP-Actin 
IWIonomers 



tl 



NUCLEUS 

Unstable 

"Trimer 



E lo ngat i on 



t 



v/ 






y R ep t* Itlve " 
y/ AcJdition ^^3: 

>T>D>a>P>Ci>p>P>lj>[j>T> 



f3^t grtrmans 






Figure 1.1. The two steps of actin filament assembly 



The S 1 subfragment has been used in electronmicroscopy to decorate actin 
filaments which confirmed kinetic studies that postulated two distinct ends to an actin 
filament. The filament ends have been defined as the plus end or barbed end (from S 1 
decoration) and the minus end or pointed end (Figure 1.1). The plus end is the more 
kinetically active end and has a K^ for ATP-actin monomers of 0.15 uM, the minus end is 



less active with a Kp of 0.5 uM. Both ends have a K^ of 6.0 uM for ADP-actin. Actin 
polymerization is a two step process (Figure 1.1) in which the initial rate limiting step, 
formation of the unstable nucleus trimer, is followed by rapid filament growth from both 
ends until the monomer concentration is depleted below the critical concentration for that 
end or the ends become capped. Once the ATP-actin monomer is incorporated into a 
filament the ATP is hydrolyzed to ADP, leaving dynamic actin filaments comprised of 
mostly ADP-actin in which the energy of hydrolysis is stored within the structure of the 
filament. The unequal kinetics of the two ends for ATP-actin allow for very precise control 
of the polymerization process, not only allowing for controlled and directed polymerization 
to take place when the appropriate signal is received, but also controlled depolymerization 
or disassembly by filament severing and/or uncapping. 
Actin Binding Proteins Important in Actin-Based Motility 

In addition to the kinetic regulation of actin polymerization, further control of 
polymerization of actin in non-muscle cells rests with the actin binding proteins. The 
function of some of these critical proteins are described briefly below: monomer 
sequestering proteins, such as thymosin B4 and profilin; capping proteins which block the 

plus end, such as villin, gelsolin, CapZ, and CapG; and severing proteins which cleave 
filaments, such as gelsolin and severin. Some of these actin binding proteins are 
responsive to calcium and phosphatidylinosititol bis-phosphate (PIP 2), and therefore 
control of polymerization is responsive to external as well as internal cell signals. 

Electron micrographs of nonmuscle cells show that actin filaments are organized 
into networks where many filaments appear to cross each other at right angles, forming an 
orthogonal mesh. The cross-linking protein ABP-280, or filamin, is responsible for 
organizing the right angle networks (Stossel, 1993). In many cell types, parallel bundles 

of actin filaments can also be seen by electron microscopy. The 105 kDa protein a-actinin 
binds to the sides of actin filaments and can link actin filaments into parallel arrays or 



bundles. The 120 kDa protein vinculin is a key component of adherins junctions and focal 
contacts, and it binds and concentrates a-actinin in this region. Vinculin has a 90 kDa head 
region containing the a-actinin binding site and a 30 kDa tail region (Price et al., 1989). The 
two regions are linked by three oligoproline sequences, the first of which is homologous 
to the Listeria surface protein ActA (see below). Protease treatment cleaves the tail from the 
head. The head region can fold over and bind to the tail, covering up many of the binding 
domains (Johnson & Craig 1995). It is likely that in the cell, association and dissociation of 
the head and tail regions serve as a regulatory switch involved in forming focal contacts and 
assembly of new actin filaments. 

Another class of actin regulatory proteins are the monomer binding proteins. Even 
under conditions expected to polymerize actin completely, unstimulated cells contain high 
concentrations of monomeric actin. In polymorphonuclear (PMN) leukocytes, for example, 
60-70% (or about 200 |xM) of the total actin exists in a monomeric form (Southwick and 
Young, 1990). It is therefore important for the cell to contain proteins which directly 
regulate actin monomer function. Thymosin B4, a 5 kDa polypeptide, may account for the 

high actin monomer concentrations in nonmuscle cells. A second monomer-binding protein 
is the 15 kDa protein, profilin. This host cell actin regulatory protein is likely to play a key 
role in bacterial actin-based motility. Profilin also binds G-actin as a one-to-one complex 
and displays a dissociation constant of 1-10 |lM. Profilin catalyzes the exchange of ADP- 
actin with unbound ATP to form ATP-actin (Mockrin & Korn, 1980, Goldschmidt- 
Clermont et al. 1991). Because ATP-actin has a higher affinity for actin filament ends, the 
facilitation of nucleotide exchange should enhance actin filament assembly. Profilin may 
also serve to enhance the affinity of the barbed ends for actin monomers and stimulate actin 
assembly by this mechanism (Carlier and Pantaloni, 1997). Finally, profilin binds to poly 
L-proline (Tanaka & Shibata, 1985), and this binding characteristic is likely to play an 
important role in concentrating the protein in locations where new actin filaments assemble 
(Southwick and Purich, 1994a). 



8 

The employment of microfilaments and another special class of actio binding 
proteins supplies the contractile mechanism of muscle cells as well as nonmuscle cells. 
These proteins are members of the myosin family. In muscle cells, myosin II forms 
multimers with moveable head domains, which by binding and release (using the energy of 
ATP hydrolysis) move actin filaments during muscle contraction. In nonmuscle cells, 
myosin I, acting as a monomer, moves microfilaments using similar contractile processes. 

As alluded to earlier, the control of actin assembly and disassembly is necessary to 
prevent unwanted polymerization and to direct polymerization and depolymerization, both 
spatially and temporally. Recently, a group of identified monomeric GTPases have been 
shown to be important in these actin regulatory processes. These GTPases, of which Ras 
is the prototype, act as molecular switches that are turned on by binding GTP and turned 
off by GTP hydrolysis. The stimulus for activation comes from a signal cascade initiated at 
a cell surface receptor. The GTPases can be linked themselves, in signal transduction 
pathways in which downstream members are stimulated to specifically activate multiple 
signal pathways (Chant and Stowers, 1995). Identified in the control of the actin 
cytoskeleton are three GTPases: Rho controls microfilament bundling to produce filopodia 
and stress fibers; Rac controls microfilament crosslinking to produce lamellipodia 
extensions; and Cdc42 acts upstream in the pathway to control Rac (Nobes and Hall, 
1995). Recently using mutation studies and genetic analysis in Caenorhabditis elegans, 
Rho was shown to be necessary in cell migration and for nerve cell axon targeting (Zipkin 
et al., 1997). It is certain other GTPases will be added to this group as more is learned 
about the regulation of the actin cytoskeleton. 
Life Cvcle and Actin-Based Motilitv of Intracellular Pathogens 

How do Shigella and Listeria gain entry into the host cell's cytoplasm and why is it 
especially important that cell-mediated immunity is critical for protecting the host against 
Listeriosis? Studies of Shigella and Listeria infection in tissue culture cells provide a new 
understanding of the stages in these infections and help to answer these questions. As 



shown in Figure 1.2, the organisms are first phagocytosed by neutrophils, enterocytes, 
macrophages (Dabiri et al. 1990), epithelial cells, and endothelial cells, as well as 
fibroblasts. Listeria produces the 80 kDa surface protein intemalin that enhances bacterial 
attachment and internalization (Gaillard et al. 1991). Performing the same function in 
Shigella are the IpaB, IpaC, and IpaD proteins (Menard et al., 1994). 

The bacterium then becomes enclosed in a subcellular compartment called the 
phagolysosome, a normally hostile and toxic environment for most bacteria. The low pH of 
this compartment, however, activates listeriolysin-O, a pore forming toxin, of Listeria 



phagocytosis 



lysis of / 

phagolysosome/ / 




Figure 1 .2. Intracellular Life-cycle of Listeria 



or a hemolysin of Shigella to lyse the phagolysosome, allowing escape of the bacteria into 
the cytoplasm. Once within the cytoplasm, the bacteria double every 60 minutes (Dabiri et 
al.,1990). They become surrounded by actin, and about two hours later, actin filaments 
begin extending from one end of the bacterium, propelling the organism through the 
cytoplasm (Dabiri et al.,1990, Tilney and Portnoy, 1989). Many bacteria subsequently 
induce the formation of filopods which are stabilized actin bundles enveloped in membrane 
resulting in elongated membrane protrusions, each containing a bacterium at their tip. These 



10 

filopods are ingested by adjacent cells, and a characteristic double membrane 
phagolysosome can be observed (Xilney and Portnoy 1989). After avoiding the hostile 
extracellular environment of immunoglobulins, complement, and extracellular antibiotics, 
the life cycle then begins over. This unusual intracellular life style helps to explain many of 
the unique clinical characteristics of Listeria and Shigella infection (Southwick and Purich, 
1996). 

A large number of experiments have shown that Shigella and Listeria use a 
mechanism of actin polymerization to move through the cytoplasm of infected host cells. 
These experiments made use of inhibitors of actin polymerization such as cytochalasin D to 
show that blocking actin assembly also blocked bacterial motility (Dabiri et al., 1990). 
Additionally, epifluorescence experiments using the fungal peptide, phalloidin, which 
preferentially binds microfilaments, conjugated to a fluorescent dye, stained actin filaments 
comprising the rocket tail of moving bacteria. Bacterial F-actin tails can also be clearly 
visualized by phase-contrast microscopy as dynamic structures that appear as rocket-like 
tails behind the moving organism. 

Mutant strains of Listeria and Shigella have been isolated or generated by 
transposon mutagenesis, in which single genes have been shown to be disrupted, and these 
strains lack actin-based motility. The IcsA gene of Shigella (Bemardini et al, 1989 and 
Goldberg et al, 1993) and the ActA gene of Listeria (Kocks et al., 1992) were identified to 
be necessary and sufficient for actin-based motility. IcsA" strains of Shigella and ActA' 
strains of Listeria are able to invade host cells, but are unable to move within the cytoplasm 
by actin assembly. ActA and IcsA have been shown to be bacterial surface proteins that are 
uniquely expressed in a polar orientation from the bacterial membrane (Goldberg et al., 
1993). 

Immunofluorescence and electron microscopy demonstrate that ActA is expressed 
on the surface of intracellular Listeria, but is not found in the actin filament rocket tails 
(Kocks et al. 1992, Niebuhr et al. 1993). The absence of ActA in the actin filament tails is 



11 

consistent with in vitro experiments which have shown that the ActA protein does not bind 
directly to actin (Kocks et al. 1992); nor does ActA stimulate the polymerization of purified 
actin (Tilney et al. 1990). Examination of the amino acid sequence of ActA reveals four 
very unusual oligoproline sequence repeats, located at residues 235- DFPPPPTDE, 269- 
FEFPPPPTDE, 304-FEFPPPPTED, and 350-DFPPIPTEE (where the number preceding 
each sequence designates the first amino acid) (Kocks et al. 1992, Domann et al. 1992). 
ActA DNA has been transfected into mammalian cells and expression of the ActA protein is 
associated with the assembly of new actin filaments (Pistor et al. 1995). All evidence 
suggests that ActA is the only Listeria protein required to stimulate actin assembly in host 
cells. In-frame deletion of the oligoproline repeats markedly reduces ActA-induced actin 
assembly, emphasizing the functional importance of these regions (Smith et al., 1996). 

There has been a growing recognition of the importance of proline rich sequences in 
mediating protein-protein regulatory interactions. When a sequence of four or more proline 
residues are bound in a polypeptide a unique extended structure is produced, the 
polyproline II helix. Much evidence suggests this extended structure of restricted mobility 
has unique and favorable binding characteristics with binding partners. Proline-rich 
sequences, because of their highly restricted mobilities, have a relatively low entropy even 
before binding; and because binding leads to a smaller drop in entropy than would occur 
for a polypeptide sequence of more flexibility, binding to proline-rich sequences is favored 
entropically. There is also evidence to suggest that binding to proline-rich sequences is 
favored enthalpically as well, because prolines form more electron-rich amide bonds. These 
two characteristics make binding to proline-rich regions more thermodynamically favorable 
and result in high affinity complexes (Williamson, 1994). Because of the high affinity of 
such interactions, proline-rich sequences are often involved in complex multiple protein 
associations. The actin-based motor utilized by Listeria for intracellular motility appears to 
be a multi-protein complex, and the proline-rich sequences found in ActA are ideally suited 
for the assembly of such a motor. 



12 



Three classes of proline-rich sequences have been defined (Williamson, 1994): (1) 
Repetitive short proline repeats [example - the (AP)6 sequence found in light chain myosin 
kinase, shown to bind to actin]; (2) Tandem repeats (example - the [SYP(P)Q(P)]5 
sequence of Dictyostelium actin-binding protein and Squid rhodopsin sequence 
[PPQYJio); and (3) Non-repetitive proline rich sequences (example - SH3 binding 
sequences XPXXPPPXP where x represents nonconserved amino acids and a hydro- 
phobic amino acid). The ActA protein falls into the second category representing a tandem 
repeat. The sequence found in each repeat is unique compared with other classes of 
prolme-rich proteins. The ActA proline repeats contain a preceding aromatic group, 
followed by a series of prolines that are flanked by a negatively charged amino acid on each 
side. This sequence now defines a specific consensus sequence found in a number of 
cytoskeletal proteins critical for actin-based motility. This consensus sequence is based on 
a register of 4-5 proline residues preceded on the N-terminal side by amino acids containing 
acidic and aromatic side chains and on the C-terminal side acidic amino acids. These 
properties define the ABM-1 (actin-based motility- 1) homology sequence: (D/F) 
FPPPPX(D/E)(D/E). ABM-1 sites are found in all four repeats of ActA, Vinculin, and 
zyxin (Purich and Southwick, 1997). 

As previously noted, the surface protein IcsA of Shigella had been identified as the 
protein required for actin-based motility by these organisms (Bemardini et al., 1989). 
Further, IcsA was demonstrated to be the only protein necessary to support bacterial 
motility in cytoplasmic extracts by stable expression of IcsA on E.coli, a bacteria that does 
not normally assemble actin (Goldberg and Theriot, 1995). Although the actin filament 
dynamics of Listeria and Shigella intracellular motility appear to be similar (our 
microinjection experiments oudined in Chapter 2 of this dissertation clearly demonstrated 
this), the protein, IcsA, shares no homologies with ActA and does not contain an ABM-1 
site now believed necessary for efficient actin assembly. It is now believed that Shigella 



13 

requires an additional host cell protein, or an ActA mammalian homologue, containing an 
ABM-1 site that is usurped during infection for assembly of Shigella's actin motor. 
A model for Actin-Based Motility of Bacterial Pathogens 

Based on evidence in the literature and our experimental findings we have 
proposed a working model for actin assembly by Listeria and Shigella. Shown in Figure 
1.3 is a cartoon of the proposed mechanism of Listeria actin assembly. This model 
describes a mechanism based on recruitment of the host cell protein, VASP, by the 
bacterium. In the case of Listeria motor assembly, the ActA protein directly binds VASP 
by its four registers of ABM-1 sequences. This binding interaction has been demonstrated 
in vivo by transfection of cDNAs coding for chimeras of ABM-1 sites, 
immunofluorescence (Bubeck et al., 1997) and solid phase binding assays (Niebuhr et al, 
1997). 

Considerable evidence suggests that VASP acts to concentrate profilin or profilin 
actin complexes at the bacteria-actin tail interface (Kang et al., 1997). VASP and profilin 
have been shown to co-localize in fibroblasts (Reinhard et al., 1995a) and profilin has been 
localized to the bacterial-actin tail interface in moving Listeria (Theriot et al., 1994). VASP 
exists as a tetramer in the cell and contains mutliple GPPPPP registers to which profilin 
binds. These oligoproline sequences are characterized by the consensus sequence 
XPPPPP (X=G, A, L, S), now known as the ABM-2 homology sequence (Purich and 
Southwick, 1997). These sequences have been found in other actin regulatory proteins 
such as the human Wiscott-Aldrich Syndrome Protein (WASP), the Drosophilia 
polypeptide, Ena, and its mammalian homolog, Mena. Because VASP as a monomer has 
four ABM-2 sequences for profilin binding and therefore, as a tetramer in the cell, has a 
potential of 16 binding sites for profilin, Kang et al. (1997) postulated that the bacteria 



14 




ADP-Aclin 
• ATP-Aclin 
A Profilin 

-W-FEFPPPPTDE 

-A- GPPPPP 

-i^-KKRRK 



Figure 1.3. Schematic model of ActAA^ASP/profilin interactions 

leading to the generation of ATP-actin monomers. Left lower 
corner depicts the bacterial cell wall containing the ActA protein 



must form an activated cluster or polymerization zone in which the necessary components, 
profilin and profilin/actin complexes, are in very high local concentrations. In addition to 
high local concentrations of polymerization competent actin, the availability of uncapped 
plus ends and G-actin turnover by actin depolymerization factor, ADF/cofilin, allows rapid 
actin polymerization to take place at the bacteria-actin filament interface, propelling the 
bacteria through the cytoplasm (Carlier and Pantaloni, 1997). 

The model for Shigella actin-based motility differs only in one component: a host 
cell adapter molecule. VASP been shown to be concentrated behind moving Shigella 
(Chakraborty et al, 1995). Our experiments (Zeile et al., 1996) in which Shigella infected 
cells were microinjected with synthetic peptides of either the ABM-1 or ABM-2 sequence at 
submicromolar concentrations, which completely inhibited bacterial motility, indicated a 
mechanism in which VASP played a central role. We demonstrated (Laine et al., 1997) 
that although no known protein of Shigella had an ABM-1 site for VASP binding, Shigella 
recruits the host cell vinculin for this purpose. Vinculin contains an ABM-1 sequence that 
is normally masked as described previously, but this site is exposed by limited proteolysis 
during the apoptotic state of the infected cell. The cleaved form of vinculin (p90) with its 



15 

unmasked VASP binding site binds IcsA of Shigella which now allows assembly of an 
actin motor identical to Listeria. 

Summary 

The purpose of the research outlined in this dissertation is to add to our 
understanding of intracellular pathogenesis by studying the exploitation of the host cell by 
bacteria. We will show in the following chapters that our contribution has been in three 
main areas. 1 ) We now appreciate that the molecular mechanism of actin motility by 
Shigella and Listeria is a shared mechanism, and that although these two bacteria are 
evolutionarily divergent they have evolved similar parasitic mechanisms. 2) We know that 
in addition to the host cell protein, actin, these bacteria exploit other key cytoskeletal host 
cell proteins during the infection process. And that in usurping critical host components, 
these bacteria have exploited different cellular processes, such as apoptosis and proteolysis 
in the case of Shigella. 3) The molecular mimicry, as revealed by these pathogenic 
organisms, instructs us about the intracellular environment in general the principles learned 
can be applied to many cellular processes. 

It is hoped that revealing the molecular mechanisms by which bacteria using actin- 
based processes to invade and spread cell to cell will aid in the discovery of more effective 
treatments for the infections caused by these organisms. In a more general sense, 
understanding the molecular basis for actin-based motility in Shigella and Listeria will give 
a better understanding of the actin cytoskeleton and the processes in which it is continually 
being remodeled in response to intracellular as well as extracellular signals. This will teach 
us about the causes of specific diseases of motile cells such as leukocytes, which become 
impaired for movement; and cancers in which cells that are not normally motile become so. 
We know these processes are actin-based--now we must understand the complex 
regulatory cascade by which these processes are controlled. 



CHAPTER 2 

RECOGNITION OF TWO CLASSES OF OLIGOPROLINE SEQUENCES IN 

PROFILIN-MEDIATED ACCELERATION OF ACTIN-BASED SHIGELLA MOTILITY 

Introduction 

In the experiments described below, we addressed the hypothesis that although 
Shigella and Listeria are evolutionarily divergent organisms they have evolved a similar 
mechanism for usurping the cytoskeleton of the host cell. We chose to test this by 
microinjecting synthetic peptides generated toward the putative binding sites of Listeria 
ActA and VASP, and microinjecting the recombinant protein, profilin, into mammalian 
cells infected with Shigella. 

Microinjection of a synthetic peptide (CFEFPPPPTDE) analogue of the second 
ActA repeat into PtK2 cells infected with Listeria rapidly and completely blocks Listeria- 
induced actin assembly at a final intracellular peptide concentration of about 80 nM 
(Southwick and Purich, 1994b). Microinjection of mosquito oostatic factor, the freely 
occurring decapeptide YDPAPPPPPP, also inhibits Listeria locomotion (Southwick and 
Purich, 1995). At similar concentrations, both of these peptides result in the loss of the 
host cell's normal actin filament structure and retraction of the peripheral membrane. 
Microinjection of a third peptide analogue DFPPPPTDEELRL derived from first 
oligoproline repeat in ActA also results in membrane retraction and loss of the normal actin 
filament architecture. These changes were associated with the dissociation of VASP from 
focal adhesion plaques and redistribution throughout the cytoplasm (Pistor et al, 1995). 
These peptides, therefore, are likely to block VASP binding to an ActA-like host protein 
(possibly vinculin) as well as block VASP binding to the oligoproline regions of ActA. 



16 



17 

In addition to binding to ActA, VASP also binds to profilin (Reinhard et al., 
1995a). Profilin is a key host cell component responsible for Listeria and Shigella 
locomotion. Depletion of profilin from Xenopus egg extracts, using beads with covalently 
bound poly-L-proHne, blocked in vitro movement of Listeria, and re-addition of profilin 
partially restored motility (Theriot et al., 1994). ProfiHn enhances the exchange of ATP on 
actin monomers (Mockrin and Korn, 1980; Goldschmidt-Clermont et al., 1991) and may 
produce higher intracellular concentrations of the more polymerization-competent ATP- 
actin at the bacterium/rocket-tail interface (Southwick and Purich, 1994b). In addition, in 
the presence of the monomer sequestering protein thymosin B4, profilin may interact with 
the barbed ends of actin filaments to lower the critical concentration for actin assembly 
(Pantaloni and earlier, 1993). 

Although the first descriptions of actin filaments being associated with intracellular 
bacteria were reported with Shigella-M&cttd cells (Bemardini et al., 1989), video 
microscopy experiments similar to those designed to explore actin-based motility in Listeria 
have not been performed in live cells infected with Shigella. Here we now performed time- 
lapse studies which reveal that Shigella moves at rates and trajectories similar to Listeria, 
suggesting these two bacteria stimulate actin based motility by similar mechanisms. 
Shigella like Listeria has an outer cell wall protein, IcsA, which is necessary for actin- 
based motility (Bemardini et al., 1989; Goldberg et al. 1993) and is sufficient to support 
actin-based movement in Xenopus egg extracts (Goldberg and Theriot, 1995). This 120 
kDa protein, however, shares no sequence identity with the Listeria ActA protein and lacks 
oligoproline sequences which might recruit host cell components to facilitate actin filament 
assembly. To test the possibility that the IcsA protein attracts a host cell oligoproline- 
containing protein to serve in place of ActA, we examined intracellular Shigella motility 
after the microinjection of two oligoproline analogues derived from ActA and VASP amino 
acid sequences. Cellular ActA analogue concentrations necessary to inhibit Listeria 
movement {i.e., in the range of 80-800 nM) blocked Shigella motility as well. The 



18 

introduction of an oligoproline peptide based on tlie VASP sequence, (GPPPPP)3, at 
considerably higher intracellular concentrations (10 ^M) also blocked Shigella movement. 
Microinjection of an equimolar binary solution of profilin with the ActA or the VASP 
analogue neutralized the inhibition of Shigella movement. Even more surprisingly, the 
binary solutions caused a 200% to 300% increase in the velocities of intracellular bacterial 
migration. These findings provide evidence for a shared mechanism involving certain 
oligoproline-containing proteins and profilin in actin-based motility of both Shigella and 
Listeria ; they also suggest that a similar mechanism may regulate actin filament assembly at 
the cytoskeleton-membrane interface of actively moving nonmuscle cells. 



Figure 2. 1 . Phase images of Shigella rocket tails. 



(A-C): Formation of a phase dense rocket tail as a Shigella bacterium migrates upward and 
to the right through a thin region of the cytoplasm in a PtK2 host cell. Images are taken at 
approximately 30 sec intervals as indicated by the time stamp. (Panel, top to bottom). (D- 
F): Formation of a phase lucent actin rocket tail as the bacterium in the lower right hand 
corner of image D migrates through the perinuclear region of a PtK2 cell. The bacterium in 
D has turned to the right in images E and F, and is migrating toward the top of the 
micrograph. A thin clear area that displaces subcellular organelles trails behind the 
bacterium and is best seen in image D. Length of time stamp bar = 12 |a,m. 



20 



A 



JK» 



14:65:46.98 



• 



D 

* 






15 


:2l:54.31^H 




■*-.,:,.„, 




14:06:15.26 



C 



14:06:43.55 



15:22:22.76 




15:22:54.50 



l 



21 
Materials and Methods 

Materials 

Peptides were synthesized by the automated Merrifield method in the University of 
Florida Protein Sequencing Core Laboratory. For microinjection the peptides were diluted 
to a stock concentration of 1-1.8 mg/mL in sterile PBS (pH 7.2) and the pH of each peptide 
solution titrated to a pH of 7.2. Bodipy-phallacidin was obtained from Molecular Probes 
(Eugene, Oregon). Primary anti-vinculin and anti-alpha actinin antibodies and fluorescein- 
conjugated anti-IgG antibodies were obtained from Sigma (St. Louis, MO). Profilin was 
purified from human platelets or from supematants of E. coli expressing recombinant 
human profilin (pET expression vector in E.coli strain BL21 kindly provided by Dr. S. 
Almo, Albert Einstein College of Medicine) using a poly-L-proline Sepharose-4B affinity 
column as previously described (Southwick and Young, 1990). 
Tissue Culture Methods and Infection Procedures 

The PtK2 cell line (derived from the kidney epithelium of the kangaroo rat Pororous 

tridactylis ) was seeded at a concentration of 1 x 10^ cells per coverslip in 35 mm culture 
dishes in 3 mL of culture media (MEM with 10% fetal calf serum, 1 % penicillin- 
streptomycin) and incubated for 72 h at 31^C and 5% C02- Shigella flexneri M90T wild- 
type strain was inoculated into brain heart infusion (Difco) and grown overnight at 37^0. 
Bacteria were harvested at mid-log phase and resuspended in MEM without antibiotics to 

give a final concentration of 1 x 10^ or a ratio of 10 bacteria per host cell. Bacteria in 3 mL 
of culture media were added to each dish followed by centrifugation at 400 x g at room 

temperature for 10 min and then incubation for 45 min at 37° C and 5% CO2. After 

incubation, extracellular bacteria were removed by washing three times with Hank's 
balanced salt (Gibco). The culture media containing gentamicin sulfate (10 Mg/mL) was 



22 

added back to prevent extracellular growth of bacteria. The monolayers were then 
incubated for 1-4 h during which microinjection and video microscopy were performed. 
Microscopy and Microinjection 

A Nikon Diaphot inverted microscope was equipped with a charge-coupled device 
camera (Dage-MTI, Michigan City, IN), and the microscope stage temperature was 
maintained at 37° C with a MS-200D perfusion microincubation system (Narishige, 
Tokyo). Digital images were obtained and processed, using an Image- 1 computer image 
analyzer (Universal Imaging, West Chester, PA). Velocities of bacterial movement were 
determined by comparing the images at two time points and measuring the distance traveled 
by each bacterium using the measure curve length function (Image I/AT program). 
Distances were calibrated using a Nikon micrometer. Differences in migration velocities 
were analyzed using the unpaired Student's t test or the Mann- Whitney nonparametric test. 
For each bacterium, velocity was determined for 3-4 time points before and 3-4 time points 
after each microinjection. One to two bacteria were analyzed for each injected cell. In each 
experiment n indicates the number of velocity measurements. Individual cells were 
microinjected with peptide using a micromanipulator and microinjector (models 5171 and 
5242; Eppendorf, Inc.) as previously described (Southwick and Purich, 1994b). 

Immunofluorescence staining using anti-a-actinin antibodies was performed as 

previously described (Dabiri et al., 1990). In experiments requiring phaUicidin staining, 
PtK2 cells were fixed with 3.7% (vol/vol) formaldehyde in phosphate-buffered saline for 
15 min at 25°C followed by treatment with 0.4% Triton X-100 and 1.7 XIO'^M bodipy- 
phallacidin (Molecular Probes, Eugene, Oregon) for 10 min at 37°C. The relative 
fluorescence intensities of the bodipy-phallacidin stained tails were measured with the 
Image- 1 system using a Genesis I image intensifier (Dage-MTI) in the linear response 
range. Gain settings were identical for both the Shigella and Listeria rocket tails. The 
relative intensity was measured at different locations on the tail with a fixed square template 



23 

(2x2 pixels, brightness function; Image-I/AT). Fluorescence intensity of an identical area 
adjacent to the actin rocket tail within the cell was measured and subtracted from each 
value. 



24 



^: * * -* 



' K 







7 



r \ \ 



,»«?■ 




•' 



Figure 2.2. Comparison of bodipy-phallacidin staining of Shigella and Listeria actin 
filament rocket tails. 



Simultaneous phase (A) and fluorescent micrographs (B) of an intracellular Shigella are 
shown. Arrows point to the bacterial-actin rocket tail interfaces. Note the faint fluorescence 
of the actin rocket tails (B) which extend from the back of many of the bacteria. The rocket 
tails are thin and demonstrate relatively low fluorescence intensity as compared to Listeria 
actin rocket tails (D). In the phase micrograph of Listeria, phase dense actin rocket tails can 
be readily visualized (C), and the tails exhibit highest bodipy-phallacidin fluorescence in the 
region nearest each bacterium (D). Infections were performed simultaneously using the 
same stock of cells and stained in parallel. Gain settings were identical for both 
fluorescence images (B, D). Bar, left lower corner of (D) = 10 |Lim. 



Figure 2.3. Simultaneous phase-contrast (A), anti-a-actinin immunofluorescence (B) and 
bodipy-phallacidin stained fluorescence (C) images of a Shigella rocket tail. 



Arrow points to the back of the bacterium which in the phase-contrast image refracts poorly 

in this region of the cell. Note the bright anti-a-actinin fluorescence as compared to that 
associated with phallacidin staining (both images were captured with gain settings in the 
linear response range of the image intensifier). Bar = 10 |lm 



26 



A 





ed Oct 26. 1994 14:29:23.5 



B 




Ued 


Oct 26, 1994 14:28:17.59 


I^^^^^B^^^^^^^^HJ^^^^^HI 


X 


;! 


Ued 


Oct 26, 1994 14:30:49.52 



27 
Results 

Characteristics of Shigella Movement and Actin Rocket-Tail Formation in PtK2 Cells 

Like Listeria, Shigella moves at relatively rapid velocities through the cytoplasm. 
Although their larger size might be expected to resist migration in a viscous medium, the 
observed mean rates of Shigella movement in PtK2 cells (0.17-0.05 |i.m/sec) were 
comparable to those of Listeria (0.15 to 0.05 fim/sec) (Southwick and Purich, 1994b; 
South wick and Purich, 1995). The maximal velocities of 0.4 /im/s&c attained by Shigella 
are rarely seen in Listeria-infccted PtK2 cells. As observed with Listeria infections, the 
mean rate of migration varied considerably from day to day. These differences appear to be 
related to the age of the tissue culture cells at the time of infection, and in all microinjection 
experiments pre- and post-treatment rates were compared in the same cells. Intracellular 
movement of Listeria in PtK2 cells is usually associated with the formation of phase-dense 
rocket-tails on phase contrast micrographs (Sanger et al., 1992; Southwick and Purich, 
1994a; 1995). On the other hand, motile Shigella are infrequently associated with phase- 
dense tails (Figure 2.1 A). Bacteria migrating in regions near or within the cell nucleus 
often display phase-lucent tails (Figure 2. 1 E). In most instances, rocket-tails are not seen 
as the bacteria move through the cytoplasm. 
Fluorescence Staining of Actin and Alpha-Actinin in Shigella Rocket-Tails 

Comparisons of bodipy-phallacidin staining of the actin filament tails reveal that 
the S/irge//a-associated structures (Figure 2.2B) have significantly lower fluorescence 
intensities than Listeria (Figures 2.2D). This observation suggests that Shigella rocket tails 
have a lower actin filament content than Listeria. As observed in Listeria (Dabiri et al. 
1990), the actin filament bundling protein and cross-linking protein a-actinin also localizes 
to the Shigella rocket tails (Figure 2.3). 



28 




Thu Feb 3, 1994 15:50:54.18 



Thu Feb 3, 1994 15:51:37.46 



Figure 2.4. Shigella movement and actin rocket tail formation in PtK2 host cells before 
and after microinjection of the synthetic ActA peptide. 



Prior to injection the bacteria are seen to move at 0. 12 |im/sec, and maximum tail length is 
6.0 |im (A, B). After injection of an estimated intracellular concentration of 80 nM of ActA 
analogue (needle concentration 0.8 |iM ActA peptide) at 160 sec, bacterial movement stops 
and the actin tails almost completely disappear (C, D). Times (indicated in sec) are mcluded 
in the lower left corner of each micrograph. The triangle (drawn by connecting three small 
phase-dense granules in the cytoplasm) served as a stable reference point. Solid bar = 10 
|im. 



29 



Arrest of Shigella Intracellular Movement by the Second Oligo-Proline Repeat Analogue in 
Listeria ActA Protein 

Bacterial motility ceases within 30 sec after injection of the ActA analogue (800 nM 

needle concentration, estimated intracellular concentration = 80 nM)(Figure 2.4 A-D). 

Phase-dense actin tails present before injection also disappear within 30 sec. Similar 

results are shown graphically in Figure 2.5A. Microinjection of this concentration of 

peptide consistently blocks Shigella movement (mean pre-injection rate of 0.06 ± 0.03 

|im/sec, SD n = 47 versus a mean post-injection rate of 0.004 ± 0.01 fim/sec, n = 85 

velocity measurements) (Table 2.1). At this low intracellular concentration the inhibitory 

effects of the ActA analogue are not always permanent (Figure 2.5A). One quarter of the 

bacteria resume migration 2-4 min after microinjection. The rates of movement, however, 

are in all instances 25-30% of the velocities measured prior to injection (0.01-0.02 

|im/sec). The inhibitory effects of the ActA are concentration dependent (Figure 2.5B). A 

lower intracellular concentration (8 nM) of ActA fails to inhibit, while higher intracellular 

concentrations (400-800 nM) consistently block intracellular movement. In some cells 

these higher concentrations also cause membrane retraction. 

Effect of Microinjecting a Binary solution of Profilin and ActA Analogue on Shigella 
Intracellular Movement 

Although high intracellular concentrations of profilin (10 |J,M, see below) can 
markedly inhibit Shigella movement, microinjection of an 80 nM intracellular concentration 
of profilin does not have a significant effect on Shigella locomotion (Table 2. 1). 
Nonetheless, microinjection of equimolar binary solutions of tiie ActA peptide analogue 
and profilin (needle concentration = 0.8-1.0 |i.M, corresponding to estimated intracellular 
concentrations of 80-100 nM) not only neutralizes the analogue's inhibition but 
significantly increases the velocities by a factor of three (mean rate of movement prior to 
microinjection 0.09 ± 0.07 |i,m/sec, n=16 vs 0.3 ± 0.1 |im/sec, n= 33 post- 
injection)(Figure 2.6A and B and Table 2.1). The differences in velocities pre- and post- 



30 

injection were highly significant on a statistical basis (p< 0.0001). Velocities increased to 
nearly 0.5 |lm/sec in some instances. 



80 nM 
ActA peptide 




TTTT. — I 



90 180 270 

Time (sec) 




.001 .01 .1 1 

ActA Peptide (jilVI) 



Figure 2.5. Effects of microinjection of the ActA analogue on Shigella motility. 



(A) Velocity of a single Shigella bacterium in a PtK2 cell before and after microinjection of 
the ActA analogue. The estimated intracellular ActA analogue concentration was 80 nM 
(needle concentration 0.8 |iM). The arrow marks the time point at which the peptide was 
introduced. The graph corresponds to the bacterium shown within triangle of the 
micrograph shown in Figure 4. (B) Effect of varying intracellular concentrations of the 
ActA analogue on Shigella intracellular velocity. Horizontal axis is in a log scale. 
Intracellular concentrations of 8 nM, 80 nM, 400 nM and 800 nM were studied. Bars 
represent the standard deviation of the mean for 30-80 velocity determinations per 
concentration. 



Introduction of the binary solution also frequently activated stationary bacteria to 
move at rapid rates (Figure 2.6B). If the stationary bacteria were included in pre- and post- 
injection velocity comparisons, the differences were also highly significant (mean pre- 
treatment velocity 0.06 ± .07 |lm/sec, n=25 vs. mean post-treatment velocity 0.25 ±0.12 
|im/sec, n=49, p< .0001). The dramatic effects of the binary solution are also illustrated in 
the time-lapse micrographs (Figure 2.6A). A bacterium can be seen to rapidly accelerate in 



31 




Figure 2.6 A Time-lapse phase micrograph of Shigella motility in a PtK2 cell before and 
after microinjection of an ActA analogue/profilin binary solution. 



(A) Time-lapse phase micrographs of Shigella motility in a PtK2 before and after 
microinjection of the ActA/profilin binary solution. This composite photograph depicts the 
path and distances covered by a single bacterium before and after microinjection of 
ActA/profilin in an equal molar ratio (estimated intracellular concentration 100 nM, needle 
concentration 1 [iM). Images show the position of the bacterium at 30 sec intervals and are 
numbered sequentially. The cell was microinjected with the binary solution between 
images 3 and 4 of the composite. Following microinjection, note the progressive increase 
in the distance traveled by the bacteria after each time interval. B) The same information in 
(A) is depicted graphically as the upper curve of Figure 2.6B. 



32 

response to microinjection of a final intracellular concentration of 100 nM of the binary 
mixture. We have found no other treatment to evoke such a marked enhancement of the 
bacterial motility. Microinjection of a lower concentration of this equimolar mixture (20 
nM) caused a statistically insignificant acceleration of Shigella velocity. (Table 2.1) 

D Analogue/ProfiJin 



o 0.5 - 


{10( 


) nM each) _, 


0.4- 

1 0.3 

=^ 0.2 


If f 


Speed, 

o o 


V 

ODQCH3CK>£HK3CHaCi 


-ft 1 - 


— 1 1 , 



150 300 450 600 

Time, sec 



Figure 2.6. B) Velocities of two bacteria in a PtK2 cell before and after the microinjection 
of an ActA analogue/profilin binary solution (100 nM intracellular concentrations of both 
reagents, shown in parenthesis; needle concentrations, 1 |iM). 



Effects of Microinjection of a VASP Oligoproline Analogue Alone and in Combination with 
Profilin on Shigella Intracellular Motility 

Introduction of the VASP analogue (GPPPPPGPPPPPGPPPPP) can also inhibit 
Shigella motility without causing significant membrane retraction (Figure 2.7A and Table 
2.1). This effect is concentration dependent (Figure 2.7B), complete inhibition being seen 
at intracellular concentrations of 10 |iM, while lower concentrations (2 and 6 |a,M) cause 
variable inhibition (note the large standard deviation bars at these two concentrations. 
Figure 2.7B). Introduction of poly-L-proline also causes a dose dependent slowing of 
bacterial velocity (Figure 2.8A). Microinjection of the same concentration of an unrelated 
peptide derived from the sequence of MAP-2 had no effect on Shigella migration (Table 



33 

2. 1). As previously observed with Listeria (Sanger et al. 1995), microinjection of profilin 
also causes a concentration dependent inhibition of Shigella movement, intracellular 
concentrations of 10 |J,M causing nearly total inhibition (Figure 2.8B and Table 2.1). 
Curiously, introduction of an intermediate intracellular concentration of profilin (6 \xM) 
resulted in a bimodal behavior. Sixty percent of the bacteria stopped moving. The 
remaining forty percent accelerated their velocity, attaining mean migration rates of 0.19 ± 
0.08 |J,m/sec (n=17). These post- injection velocities were significantly higher than the 
bacteria's preinjection velocities of 0.14 ± 0.05 (n=28, p = 0.039). 



^ A VASP Analogue (10 \m) R 




-"■ — ■ — r 

90 180 270 

Time (sec) 




2 4 6 8 10 

VASP Peptide (\xM) 



Figure 2.7. The effect of microinjection of VASP peptide analogue on Shigella motility in 
a PtK2 cell. 



A) Microinjection of VASP peptide analogue, 10 |lM intracellular concentration, into 
Shigella infected PtK2 cell. The Arrow represents the approximate time of the 
microinjection. (B) Effect of varying intracellular concentrations of the VASP analogue on 
Shigella intracellular velocity. The estimated intracellular concentrations of the 
microinjected peptide are plotted on the horizontal axis. Bars represent the standard 
deviation of the mean for n = 20-40 velocity measurements per concentration. 



34 

The effect of microinjecting a binaiy mixture of profilin and the VASP oUgoprohne 
analogue was also examined (Figure 2.8C). In vitro experiments employing profilin 
tryptophan fluorescence have recently demonstrated that the (GPPPPP)3 peptide binds to 

profilin with a Kd in the 10-5 M range (Kang et al., 1997). Based on these findings, high 
equimolar concentrations (10 |i,M intracellular concentrations) of both profilin and the 
VASP oligoproline analogue when microinjected (baring interference from other 
intracellular constituents) should exist as a complex in the cell. We predicted that such a 
complex might neutralize the inhibitory activity of the two components. In fact, 
microinjection of this binary mixture accelerates Shigella movement, velocities increasing 
by a mean of 200% (pre-injection mean velocity: 0.09 ± 0.05 )lm/sec, n = 25 vs. post- 
injection mean velocity: 0.18 + 0.10, n=61, p< 0.0001) (Table 2.1). Introduction of an 
equivalent binary mixture of poly-L-proline and profilin inhibits Shigella movement (Figure 
2.8D and Table 2.1). Microinjection of a lower equimolar concentration of the VASP 
analogue and profilin (1 |lM intracellular concentrations) fails to accelerate Shigella 
migration (Table 2.1). 



35 




o.o 




O 1 6 1 O 1 4 
PLP (i-lM) 



O.I 



o.o 



O 1 2 6 1 O 

Profilin (illM) 



o 
a> 



o 
O 

CD 



0.5 - 
0.4 - 
0.3 
0.2 

0.1 
O.O 



VASP Analogue/ 
Profilin 
(10 (j^M eacl-i) 



PLP/Profilin 
(2.5 |j,IVI/10 p.M) 




0.1 



O.O 



180 360 

Time (sec) 




90 180 270 

Time (sec) 



Figure 2.8. Microinjection of poly-L-proline, profilin and mixtures of VASP analogue and 
profilin and mixtures of poly-L-proline and profilin on Shigella motility. 



Dose dependence of (A) poly-L-proline and (B) profilin inhibition of Shigella intracellular 
motility. The mean velocities of Shigella intracellular migration in PtK2 cells are shown 
following the microinjection of increasing intracellular concentrations of the two 
polypeptides. Each point represents the mean of 20-40 velocity measurements. 
Introduction of an estimated intracellular concentration of 6 |aM profilin (needle 
concentration 60 |J,M) resulted in a bimodal behavior, 40% of the bacteria accelerating their 
velocity while 60% stopped moving (see results). 



(C) The velocities of a bacterium migrating through a PtK2 cell before and after the 
microinjection of a VASP analogue/profilin binary solution and (D) before and after the 
microinjection of binary solution of poly-L-proline and profilin. The values in parenthesis 
are the estimated intracellular concentrations of the two reagents. Vertical arrows indicate 
the time when each solution was injected. These individual experiments are representative 
of numerous experiments for each condition (see results and Table 2.1) 



36 



Table 2. 1 Effects of Microinjected Peptides on Shigella Intracellular Motility. 

Additions Intracellular Pre-injection Post-injection Post-injection/ P value 

Concentration Velocity Velocity Pre-injection Velocity 

(mean, |j,m/sec, SD) 

Act A peptide 80 nM 0.06 ± 0.03 0.004 ± 0.01 0.07 < 0.001 

CFEFPPPPTDE (n=47) (n=85) 

Profiiin 80 nM 0.14 ± 0.04 0.12 ± 0.06 0.85 N.S.' 

(n=16) (n=21) 

ActA peptide 80 nM/ 0.09 ± 0.07 0.30 + 0.11 3.33 < 0.001 

and Profiiin 80 nM (n = 16) (n=33) 

20 nM/ 0.13 ±0.05 0.17 ± 0.08 1.31 N.S. 

20 nM (n=15) (n=12) 

VASP peptide 10 (iM 0.13 + 0.05 0.02 + 0.05 0.15 < 0.001 

(GPPPPP)3 {n=40) (n=65) 

Profiiin 10 nM 0.07 ± 0.03 0.02 ± 0.05 0.28 < 0.001 

(n=31) (n=21) 

VASP peptide 10 \iW 0.09 ± 0.06 0.18 ± 0.10 2.00 = 0.002 

and profiiin 10|iM {n=25) {n=61) 

1 M-M/ 0.12 ± 0.06 0.07 ± 0.04 0.58 N.S. 

1 nM {n=6) {n=16) 

Poly-L-proline 2.5 ^M/ 0.14 ± 0.08 0.06 ±0.11 0.43 < 0.001 

and profiiin 10 |iM (n=29) (n=45) 

MAP-2 peptide 10 (iM 0.15 ± 0.05 0.15 ± 0.07 1.00 N.S. 

VKSKIGSTDNIKYZPKGG (n = 17) (n=44) 

* N.S. = not significant 



Discussion 

Dynamic remodeling of the actin cytoskeleton must be controlled (Stossel, 1993; 
Condeelis, 1993), and bacterial pathogens must utilize these regulatory processes to 
achieve actin-based motility in host cells in their efforts to evade host defense mechanisms. 
We compared mechanisms underlying Listeria and Shigella movement in PtK2 host cells. 
While Shigella rocket tails have a lower F-actin content than Listeria, the average velocities 
of both pathogens are quite similar. As observed with Listeria, we now find that Shigella 

rocket-tails also contain the actin bundling and cross-linking protein a-actinin shown to be 



37 

critical for Listeria motility (Dold et al., 1994). These similarities raised the possibility that 
these two distinct pathogens may be adopting convergent mechanisms to subvert the host 
cell's actin regulatory system to allow their locomotion within cells and their spread from 
cell to cell. To explore this possibility, the inhibitory effects of oligoproline peptides based 
on the sequences in the ActA protein and VASP were examined in cells infected with 
Shigella. Over the same concentration range that inhibited Listeria intracellular motility 
(Southwick & Purich, 1994b), the ActA analogue likewise blocked Shigella movement. 

We originally hypothesized that the ActA analogue acted by competitively inhibiting 
profilin binding to bacterial cell wall ActA protein; however, in vitro experiments failed to 
demonstrate any binding of the ActA oligoproline analogue to profilin (Kang et al., 1997). 
The discovery that a second host cell actin regulatory protein VASP may serve to link 
profilin to ActA now provides a self-consistent explanation for our results (Reinhard et al. 
1995). It is likely that the ActA oligoproline analogue FEFPPPPTDE dissociates VASP 
from both Listeria and Shigella. Based on the estimates of Reinhard et al. (1992), the 
concentration of VASP tetramer in platelets is approximately 0.5-1 (iM. The content of 
VASP in other cells is considerably lower (i.e., approximately 100 nM). The latter value is 
quite close to the estimated intracellular concentrations of ActA analogue (80 nM) found to 
arrest Shigella motility. It is noteworthy that Listeria intracellular movement is inhibited by 
both the ActA analogue and oostatic factor in the identical concentration range. This 
behavior would be predicted if the peptides interact directly with the limited intracellular 
pool of VASP. 

Dissociation of VASP from the surface of the bacteria would be expected to prevent 
the concentration of profilin at the bacterial-actin tail interface blocking further actin 
assembly at this site, thereby preventing bacterial movement (Figure 2.8). Based on our 
recent studies demonstrating that profilin binds directly to a contiguous triad of GPPPPP 
repeats spanning positions 172-189 in VASP (Kang et al, 1997), we predicted that 
microinjection of a synthetic peptide containing this 18 residue triad would block profilin 



38 

localization at the bacterial actin interface and prevent bacterial induced actin filament 
assembly and intracellular movement. Our experiments confirmed this expectation. The 
intracellular concentrations of peptide required to achieve inhibition of motility were 
considerably higher than the ActA analogue (10 )iM GPPPPPGPPPPPGPPPPP versus 80 
nM FEFPPPPTDE), reflecting the higher concentrations of profilin likely to be present in 
PtK2 cells as compared to VASP and/or a lov^^er affinity of profilin for VASP oligoproline 
sequence. It is of interest that other investigators have recently demonstrated that the same 
VASP analogue can dissociate profilin from VASP in vitro (Reinhard et al. 1995), 
providing further biochemical support for our inferences about the mechanism of action of 
the VASP analogue in Shigella infected cells. We also find that this same VASP analogue 
inhibits Listeria intracellular movement at identical concentrations (Kang et al., 1997). 
Therefore both Shigella and Listeria are likely to utilize VASP and profilin to induce actin 
assembly in host cells. While all of our results are consistent with the above interpretation, 
these synthetic peptides may not be entirely specific for the proposed targets, and impaired 
bacterial movement could represent a nonspecific side effect. Other of our findings argue 
against such an interpretation. First, introduction of high intracellular concentrations of an 
unrelated peptide fail to impair motility, excluding a nonspecific toxic effect of synthetic 
peptides. Second, the ability of equimolar concentrations of profilin to totally reverse the 
inhibitory effects of the peptides suggests specific protein-protein interactions are 
responsible for the observed inhibitory effects. Our observations, however, do not exclude 
the possibility that other host cell actin regulatory proteins in addition to VASP and profilin 
may play roles in Listeria and Shigella intracellular motility. 

What then can be said about the result of our experiments with binary solutions 
containing profilin and either of the aforementioned oligoproline sequences? Simultaneous 
introduction of a profilin and ActA analogue or profilin and the VASP analogue binary 
solution did more than simply neutralize the inhibitory action. In fact, we were surprised to 
find that co-injection actually stimulated Shigella to move at rates that were two to three 



39 

times greater than their usual velocities. Introduction of the binary solutions even 
occasionally caused previously quiescent bacteria to commence moving, and these bacteria 
often reached maximal velocity. This stimulation of movement was observed following the 
addition of only 80-100 nM concentrations of profilin and the ActA analogue, the same 
concentration range where microinjection of ActA analogue alone evoked maximal 
inhibition of both Listeria and Shigella movement. Binding experiments monitoring 
tryptophan fluorescence of profilin fail to detect binding of the ActA analogue to profilin at 
concentrations of 100 )lM (Kang et al., 1997). Therefore, it is unlikely that these two 
polypeptides alone form a binary complex before or after microinjection into the cell. They 
are more likely to form a ternary complex with a third host cell protein, possibly VASP, 
and this complex in turn could stimulate actin assembly. In vitro binding experiments 
indicate that the VASP analogue and profilin will associate at the concentrations used in our 
experiments (lO-^M range, Kang et al., 1997). Therefore, the acceleration of Shigella 
motility by the binary mixtures of VASP and profilin suggests that the profilin-VASP 
complex can enhance actin assembly in nonmuscle cells. Although further experiments will 
be required to fully characterize these interactions, the present studies do indicate that under 
the appropriate conditions profilin can stimulate actin assembly. 

Based on our current findings, a working model of how Shigella induces actin 
assembly in host cells can be constructed (Figure 2.9). Because the IcsA surface protein of 
Shigella possesses no ActA oligoproline VASP binding sequence, IcsA protein probably 
attracts a host cell VASP-binding protein to the bacterial surface to concentrate VASP 
which in turn binds profilin. Profilin stimulates actin filament assembly behind the 
bacterium, and this polymerization process propels the bacterium through the host cell 
cytoplasm. The mechanism(s) by which profilin stimulates actin assembly in cells 
remain(s) ill-defined. In the presence of the monomer sequestering protein, thymosin 64, 
profilin can lower the critical concentration of actin filaments (Pantaloni & Carlier, 1993). 
Profilin also enhances nucleotide exchange on actin monomers (Mochrin & Kom, 1980; 



40 

Goldschmidt-Clermont et al., 1991). Under the rapid assembly conditions, 40-200 
monomers per sec, associated with Shigella locomotion at rates of 0.1-0.5 jim/sec, 
nucleotide exchange could prove to be the rate limiting step for new actin assembly and 
profilin could serve to accelerate this process. In the present model we have illustrated 
ATP - ADP exchange on actin monomers as the most likely explanation for profilin's 
ability to stimulate host cell actin assembly. While additional biochemical experiments 
promise a rigorous test of this scheme, a key finding in support of the model is the recent 
immunofluorescence study demonstrating VASP localization on intracellular Shigella 
(Chakraborty et al., 1995). 

The observation that the ActA analogue can block both Shigella and Listeria actin- 
based motility suggests that Shigella probably recruits to its surface a host cell protein that 
contains an ActA-like oligoproline sequence. Kaduragamuwa et al. (1993) suggested that 
vinculin, itself an oligoproline-containing actin-binding protein, might serve in place of 
ActA in Shigella actin-based motility. When Shigella infects host cells, vinculin is lost 
from focal adhesion plaques and could be concentrated on the bacterial surface. Although 
we clearly observed immunolocalization of vinculin at focal adhesion contacts, we could 
not demonstrate any accumulation of this protein on the cell wall of intracellular Shigella 
(data not shown). Such observations do not completely exclude vinculin as the candidate 
ActA-like host protein, because the amount of vinculin needed on the bacterial surface may 
be below our detection limit. Alternatively, yet another oligoproline-containing host cell 
protein may fulfill the requirement for an oligoproline recognition site. Determining the 
identity of this protein will be of great interest because this ActA-homologue is likely to 
play a key role in the generation of new actin filaments required for the extension of 
lamellipods and pseudopods in nonmuscle cells. 

In conclusion, our finding that the ActA analogue arrests Shigella motility indicates 
that its locomotion requires the presence of an oligoproline-containing protein that binds to 
the bacterium's surface in a manner mimicking the action of Listeria ActA protein. 



41 

Moreover, we have demonstrated for the first time that microinjection of a mixture of 
profilin and the ActA sequence FEFPPPPTDE (or the GPPPPP triad from VASP) can 
markedly accelerate actin-based motihty in Uving cells. This represents an unprecedented 
finding that factors introduced by microinjection can actually stimulate directional 
intracellular actin assembly. These in vivo experiments emphasize the importance of a 
discrete active pool of profilin that is likely to be responsible for stimulating new actin 
filament assembly. Shigella and Listeria, two bacterial pathogens with structurally 
unrelated membrane surface proteins, have thus managed to subvert the host's contractile 
system to generate force needed for intracellular movement, an evolutionary achievement 
that allows these pathogens to spread from cell to cell and cause disease. This same system 
is likely to play a role in promoting localized actin assembly necessary for dynamic 
remodeling of the leading edge during chemotaxis and phagocytosis. 



42 



Actin-Based Locomotory Unit 
in Shigella flexneri 



T/ d; D 



Host Cell 

ActA 
Homologue 



T) D) 



Profilin 




IcsA C< r 'T^ VASf 



I 



AOP + )ATP 



t; d 



bBcterial r ^ 

cell wall ATP+ )ADP 



t; d; d 



Figure 2.9. Working model showing the primary components likely to be involved in the 
actin-based locomotory unit of Shigella. 



Shigella contains on its surface the 1 20 kDa protein IcsA that is likely to attract an ActA-like 
mammalian protein homologue onto the bacterial surface. This ActA-like protein contains 
one or more VASP binding sequences (designated as a hatched region) responsible for 
attracting VASP to the bacterial surface. Because of tetrameric structure, VASP is capable 
of binding up to 16 profilin molecules, serving to highly concentrate profilin at the 
bacterial-actin tail interface. Profilin may promote actin filament assembly by increasing 
the rate of ADP-ATP exchange on actin monomers (chevrons) or profilin may usher actin 
subunits onto the barbed ends of actin filaments. Microinjection of the ActA peptide 
FEFPPPPTDE is thought to disrupt VASP binding to the ActA homologue on Shigella and 
microinjection of the VASP peptide (GPPPPP)3 would be expected to dissociate profilin 
from VASP. Both peptides act at different steps in Shigella-'mdnccd actin assembly and 
disperse locomotory elements (VASP and/or profilin) from the bacterial surface, thereby 
blocking actin rocket tail formation and bacterial motility. 



CHAPTER 3 
VINCULIN PROTEOLYSIS UNMASKS AN ACTA HOMOLOGUE FOR ACTIN- 

BASED SHIGELLA MOTILITY 

Introduction 



In our recent work (see chapter 2) and work by others (Bernardini et al., 1989; 
Dabiri et al., 1990; Heinzen et al., 1993; Cudmore et al., 1995) it had become increasingly 
clear that the microbial pathogens Listeria monocytogenes, Shigella flexneri, rickettsia, and 
vaccinia virus share some of the same components for actin-based motility. Our second 
aim of this research was to identify the component or components supplied by the host cell 
during assembly of the actin motor of Shigella. Identifying this component would be of 
great value in describing the general mechanism of actin assembly that has been conserved 
in cellular processes of eukaryotic cells. 

We hypothesized that Shigella must recruit a Listeria ActA-like molecule or ActA 
mammalian homologue during its actin-based intracellular spread in order to supply a 
docking site for VASP. This adapter molecule according to the hypothesis would have an 
ABM-1 sequence necessary for VASP binding and would also have a domain for binding 
the surface protein, IcsA of Shigella. To isolate this molecule we would employ a number 
of approaches based on these unique characteristics. 

The first approach probed for the ActA homologue based on its affinity for IcsA. 
We would generate an affinity column with purified recombinant IcsA covalently bound to 
N-hydroxysuccinimide-activated sepharose and screen cell lysates for binding partners. A 
second solid phase assay would be employed in which purified radiolabeled recombinant 
IcsA would be used to probe cell lysates immobilized to a solid support. Proteins identified 
by these screens would be submitted for sequence analysis. 



43 



44 

Shigella infection had been shown to deplete vincuhn from the focal contacts of 
host cells (Kaduragamuwa et al., 1991), and IcsA is known to bind vinculin and to 
concentrate vinculin to the back of intracellular bacteria (Suzuki et al. 1996). Vinculin also 
has an ABM-1 sequence, EPDFPPPPPDLE, which might be recognized by an antibody 
(FS-1) that we had generated against the synthetic ActA peptide, FEFPPPTDE. The second 
approach was to demonstrate, by co-localization experiments using immunofluorescence, 
that the FS-1 antibody recognized an epitope on the rear-ward pole of a moving bacteria 
and in addition, at the same interface, vinculin could also be localized using a anti-vinculin 
monoclonal antibody. 

Further experimental evidence was gathered by microinjection of Shigella infected 
PtK2 cells with the FS-1 antibody and a synthetic peptide, Vinc-1, of the sequence, 
EPDFPPPPPDLE, from the first oligoproline repeat of vinculin. It was hypothesized that 
either of these moieties could uncouple Shigella's actin motor and this would be further 
evidence for involvement of vinculin as the VASP binding adapter molecule in Shigella 
actin-based motility. 

Concurrent to my work, Dr. Ron Laine using the FS-1 antibody discovered that 
platelet extracts contained one or more cross-reactive proteins. A major protein band 
separated by isoelectric focusing and identified by westem blotting was excised and 
submitted for sequencing and this was identified as a 90 kDa vinculin head-fragment, 
which retains after cleavage, an ABM-1 sequence at its carboxy-terminus. 

Our data suggested that Shigella infection results in the proteolysis of intact 120 
kDa vinculin, thereby generating a p90 polypeptide that specifically binds to IcsA and 
concentrates on the bacterial surface. Infection by Shigella initiates the apoptotic cascade 
(Zychlinsky et al., 1 996) in which cellular proteases are activated. It was reasonable to 
expect that vinculin might be a target for one of these proteases which could release it from 
the focal contacts to be available to the bacteria, as p90 with its unmasked ABM- 1 site. To 



45 

test this, we designed an experiment to analyze whole cell lysates of PtK2 cells for cleavage 
of full length vincuhn to the p90 adapter molecule during infection. 

Additional evidence for the role of vinculin p90 as the adapter molecule in the 
Shigella actin motor was obtained by microinjection experiments designed and implemented 
by Dr. F.S. Southwick. Human platelet profilin was purified from platelets by Dr. Fan 
Kang and aliquots were proteolyzed by thermolysin digestion and the p90 vinculin head 
and p30 vinculin tail were separated and purified by FPLC. Microinjection of the p90 
polypeptide, but not intact vincuhn, into 5'/zige//a-infected PtK2 cells accelerated 
intracellular motility of the bacteria by a factor of three. 

Our findings indicate that the 90 kDa head-fragment of vinculin can serve as the 
ActA homologue required for Shigella actin-based motility, and vinculin proteolysis is 
likely to serve as a molecular switch that unmasks this protein's ABM-1 oligoproline 
sequence to bind VASP on the bacterial surface and to promote the assembly of an actin- 
based motor. 



Figure 3.1. Characteristics of anti-ActA peptide antibody: Immuno-localization, inhibition 
of motility, and identification of human platelet p90 polypeptide. 



(A) Fluorescence image of S'/i/geZ/a-infected PtK2 using bodipy-phalloidin to label 
polymerized actin. The thin white bars demarcate the interface between the bacterium and 
the trailing actin rocket tail. Bar = 10 |lm 

(B) Phase-contrast image of the same field shown in Panel A. 

(C) Indirect immunofluorescence image of the same cells using the FS-1 antibody raised 
against the FEFPPPPTDE sequence in Listeria ActA protein. 

(D) Speed measurements of Shigella in PtK2 cells before and after microinjection of FS-1 
anti-ActA-peptide antibody. The dashed line indicates the time of microinjection of 
antibody (40 nM calculated intracellular concentration; needle concentration 0.4 |iM). Bars 
represent the standard error of the mean (SEM) of 13 different bacteria at each time point. 
In order to compare different bacteria, values were graphed as the ratio V(t)A^(0), where 
V(t) is the velocity at each time point and V(0) is the initial velocity V(0) at t = sec. 
Comparisons of actual pre- and post-injection speeds also demonstrated a highly significant 
inhibition of Shigella motility following introduction of the FS-1 antibody, (mean pre- 
injection speed: 0.1 1 ± 0.01 |im/sec SEM n = 46 vs. post-injection: 0.02 ± 0.01 |im/sec n 
= 48, p<0.0001). This same concentration of antibody also significantly inhibited Listeria 
intracellular motility (mean pre-injection velocity 0.1 1 ± 0.01 |im/sec n = 14 vs. 0.02 ± 
0.01 |im/secn= 18, p<.0001). 

(E) Ponseau-S staining of an electroblot of a two-dimensional isoelectric focusing/SDS 
electrophoresis gel of platelet membrane extracts. The boxed area shows the major spot 
identified by FS-1 antibody (raised against ActA peptide). (from Laine et al., 1997) 

(F) Same electroblot stained with the FS-1 antibody. Two major cross-reactive 
polypeptides are identified: the 90 kDa polypeptide selected for microsequencing, and a 53 
kDa polypeptide, (from Laine et al., 1997) 



47 




Anti-ActA 

antibody (FS-1 

40 nM intracellular 




kDa 

97 — 
66 — 

45 — 



kDa 



60 120 

Time, sec 



II- tr ■ 



• *~ ■• 



■+- — ■- f^rs^ 



Ponseau-S 




Anti-ActA-peptide 



48 



Materials and Methods 

Materials 

PtK2 kangaroo rat kidney cells were grown and infected with Shigella flexneri 
strain M90T, serotype 5, or Listeria monocytogenes 10403S, virulent strain serotype- 1, as 

previously described (Dabiri et al., 1990; Zeile et al., 1996). Poly-L-proline (MW^ygj.^ g 

= 5600), aprotinin, leupeptin, pepstatin A, PMSF, and DTT were obtained from Sigma 
Chemical Co. (St. Louis, MO). NBD-Bodipy-phalloidin was purchased from Molecular 
Probes, Inc. (Eugene, Oregon). ActA peptide (CFEFPPPPTDE) and vinculin Vinc-1 
peptide (PDFPPPPPDL) were synthesized and HPLC-purified in the University of Florida 
Protein Sequencing Core Lab. 

Antibodies 

\ 

i 

Anti-vinculin 1 1-5 mouse monoclonal antibody (Vin 1 1-5) was obtained from ? 

i 
Sigma (St Louis, MO). The rabbit serum containing anti-VASP polyclonal antibody was a I 

kind gift of Dr. Ulrich Walter (Medical University Clinic, Wurzburg, Germany). f 

Polyclonal rabbit anti-ActA-peptide antibody (FS-1) was raised by immunization with a i 

peptide (CFEFPPPPTDE), corresponding to the ActA's second oligoproline repeat | 

(Southwick and Purich, 1994b) and coupled to keyhole limpet hemocyanin (Cocalico ( 

Biologicals, Reamstown, PA). Monospecific IgG was then isolated by immuno-affinity ; 

chromatography on CFEFPPPPTDE peptide coupled to cyanogen bromide-activated 

Sepharose 4B (Laine et al., 1988; Laine and Esser, 1989). Commercial secondary 

antibodies conjugated to rhodamine, fluorescein, alkaline phosphatase, or horse radish 

peroxidase were used without further purification. 



49 

Purification of Recombinant IcsA 

Recombinant Shigella IcsA was cloned from isolated virulence plasmid (Casse et 
al., 1979) of Shigella by PCR using forward primer 5'-CTG ATA ATA TAG CAT ATG 
AAT CAA ATT CAC-3' and reverse primer 5'-CAA GCT GTG AAC TAG GAT CCC 
GAG TAG TCA-3' . After digestion with Nde I and BamHI, the PCR product was 
subcloned into pET15b expression vector(Novagen, Inc., Madison, WI.) which would 
upon expression generate a fusion protein of IcsA with 6 N-terminal histidines. Bacterial 
strain BL21(DE3)(Novagen, Inc) was transformed with the expression vector 
pET15b/IcsA-histag. Correct sequence of the insert was confirmed by DNA sequencing 
performed by the DNA Sequencing Core facility at the University of Florida. IcsA-histag 
protein was expressed by induction of the transformed strain at an AgQQ=0.6 to 0.4 mM 
IPTG final concentration and growth for 3 h post induction. The bacteria were harvested 
by centrifugation at 4400 xg for 10 min at 4°C and the cell pellet frozen in liquid nitrogen. 
The cell pellet was thawed and resuspended in solubilization buffer, 0.02M Tris-HCl, pH 
7.9, 0.5 M NaCl, 6 M Urea and the solution mixed for 1 h at room temperature, then 
sonicated to shear the DNA. The solution was centrifuged at 27,000 xg for 30 min at 4°C. 
The recovered supernatant was filtered and then applied to Pharmacia 1 6/60 G-200 
Superdex gel filtration column equilibrated in solubilization buffer at a flow rate of 0.5 
mL/min. Pooled gel filtration column fractions were then applied to 5 mL bed volume 
Ni^"^-chelating sepharose column (Chelating Fast Flow Sepharose from Pharmacia) charged 
with 100 mM NiS04 and equilibrated in solubilization buffer. The column was washed 
and bound proteins were eluted with a gradient from 20 mM to 500 mM imidazole. 
Column fractions were collected, analyzed by SDS-PAGE, and pooled. Pooled fraction of 
IcsA were refolded by step dialysis in steps from 6 M to 4 M to 2M to 1 M to 0.5 M to 
Urea over 36 h at 4°C. Protein concentrations were determined by Bio-Rad protein assay. 
From 1 liter of bacterial culture, 5 mg of purified IcsA was obtained. 



50 



Affinity Chromatography 

From PtK2 cells grown on 150 mm plates, subcellular fractions were prepared by 
differential centrifugation, a cytosolic fraction (100,000 xg supernatant) and a membrane 
fraction (100,000 xg pellet). An IcsA affinity column was prepared by binding purified 
IcsA to a HiTrap NHS-activated 1 mL affinity column (Pharmacia Biotech,Uppsala, 
Sweden) according to manufacturer's instructions with a calculated 95% binding 
efficiency. Subcellular fractions were applied to the column equilibrated in 0.1 M NaPi, 
pH 7.2, 0.05 M NaCl at a flow rate of 0. 1 mL/min. After washing, bound proteins were 
eluted in high salt at a flow rate of 0.2 mL/min, and fractions collected for analysis by 
SDS-PAGE and western blotting. 
Solid Phase Overlay Assay 

The Shigella protein, IcsA was ''''S-labeled by metabolically labeling growing E. 
coli expression cells containing the pET15b/IcsA-histag vector. Methionine and cysteine 
radiolabeUng was accomplished by adding Trans ""^S-label (methionine/cysteine) from ICN 
Pharmaceuticals, Inc. (Irvine, Ca.) to the growing cultures. Purification of the histidine 
tagged fusion protein was done as above, except the gel filtration step was omitted. 
Purified IcsA-histag was used in overlay assays in a concentration of 66 ug/mL, specific 
activity 0.13 uCi/mg to 1.23 mg/mL, specific activity 0.68 uCi/mg. Subcellular fractions 
of mammalian cells were prepared as above, assayed for total protein concentration, 
separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF)(Bio-Rad, 
Hercules, Ca.) membrane using electroblotting by tank transfer in 0.01 M MES, pH 6, 
20% methanol overnight at 4°C, 35 volts (constant voltage). Additional lanes on the blot 
included purified full length vinculin and the cleaved p90 form. After transfer the 
membrane blots were then incubated in blocking buffer; 5% milk, TBS (0.1 M Tris-HCl, 
pH 8.0, 0.15 M NaCl), 0.1% NaN, for 1 h to eliminate non-specific binding and then 



i 51 



incubated with radiolabeled IcsA in blocking buffer overnight at room temperature with 

gentle shaking. The blots were then washed 2 min for a total of three washes in TBS. The 

membranes were dried and exposed to X-ray film (Kodak X-OMAT AR) overnight at - 

70°C. 

Microinjection Experiments 

Individual PtK2 cells were microinjected with the antibody and peptides and 
velocities were measured and data analyzed as previously described in chapter 2 
(Southwick and Purich, 1994b; Southwick and Purich, 1995; Zeile et al., 1996). 
Vinculin Proteolysis after Shigella Infection of PtK2 Cells 

To quantitate the p90 content of PtK2 cells, 150 mm culture dishes containing 
semiconfluent PtK2 cells were infected with Shigella as described above. At various times 
(Ih, 2h and 3h) after initiating infection, cells were harvested into a cocktail of protease 

inhibitors (2 |ig/mL aprotinin, 1 |J,g/mL leupeptin, 1 |ig/mL pepstatin A, 1 mM PMSF, 1 

mM EOT A, 1 mM EDTA, and 1 mM DTT in 0.1% Triton X-100). The cells were mildly 
sonicated at a power setting of 2, 80% duty cycle for 1 min and then centrifuged at 27,000 
xg for 30 min at 4°C. The samples were concentrated with a Centriprep-10 membrane 
concentrator (Amicon Corporation), standardized with respect to protein concentration and 
then suspended in 2 x SDS sample buffer, boiled for 5 min, and subjected to SDS-PAGE 
and transferred to PVDF membrane as described above. Uninfected cells and cells exposed 
to E. coli were extracted and processed in an identical manner. For western blot analysis, 
membranes were incubated in blocking buffer; phosphate-buffered saline (0.137 NaCl, 
0.003 M KCl, 0.0043 M NajHPO^, 0.0014 KHjPO^, pH 7.3), 1% BSA, 0.05 % Tween- 
20 during the blocking and antibody binding steps. Primary antibody anti-vinculin clone 
1 1-5 (Sigma) and a-actinin (Sigma) were used at a dilution of 1:1000 and the polyclonal 
FS-1 anti-ActA was used at a dilution of 1:500. Secondary antibodies used included goat 
anti-rabbit IgG and rabbit anti-mouse IgG conjugated to horse radish peroxidase. Bound 



52 

secondary antibody was visualized using tiie SuperSignal enlianced chemiluminescence 
metliod (Pierce, Rockford, IL.). Membrane blots were reprobed by stripping with strip 
buffer; 2 % SDS, 0.0625 M Tris-HCl, pH 6.8, 0.1 M b-mercaptoethanol at 60°C for 30 
min, washing with PBS and then blocking and repeating with new antibody as above. 

Indirect Immunofluorescence Microscopy 

PtK2 cells were infected with Shigella as described in chapter 2. When it was 
determined by microscope, that the majority of the bacteria were moving by actin assembly, 
the cells were fixed in 3.7 % formalin in a standard salt solution (0.1 M KCl, 0.01 M KPi, 
0.001 M MgClj pH 7.0), and permeabilized with 0.2 % (v/v) Triton X-100. Different sets 
of the fixed and permeabilized cells were incubated with different primary antibodies; 
monoclonal anti-vinculin clone 1 1-5 at 1:200 dilution, monoclonal anti-vinculin hVin-1 at 
1:200 dilution, polyclonal FS-1 at 1:50 dilution, polyclonal vinculin tail antibody (Dr. S. 
Craig, Johns Hopkins University) at 1:500 dilution, in blocking buffer (10% BSA, 
standard salt, pH 7) for 1 h at 37°C. The cells were washed 2 times with blocking buffer 
and then secondary antibody was added at 1:200 dilution and incubated for 1 h at 37''C. 
Secondary antibodies used were; anti-mouse or anti-rabbit IgG conjugated to fluorescein 
isothiocyanate (FITC), or tetramethylrhodamine isothiocyanate (TRITC). F-actin was 
stained with either FITC or TRITC conjugated phalloidin added to the cells after washing to 
a final concentration of 0.2 uM. The cells were then washed 3 times and Fluoromount-G 
(Fisher), an anti-quenching preservative, was placed on the cell surface and then the cell 
layer sealed with a glass coverslip. Microscopy and image analysis was as previously 
described in chapter 2. 



53 



f iRcuiin: General Stri 






Jill 




Blnaingt Sites 



VIN1 1-5 Antibody 
Binding Site 



,:^ 



St8a.v.age-, 



TZv^-vvZ^ 



'r-Actl 


iitrg Sites ' ,, 


\ ~^ 




^i>r^> 


: \ \ '-. \ \ \ \\'^ \ \ '•.fi^ 



''Viae -2 



ABM-lSlt» 

Vinc-1 84:0 -P»Pl?FPl?PI>E.:-e49 

--,FSPPP-D- 
&ctA-2 26S-fSF?PPPTDS-27 8 



Figure 3.2. Structural organization of human vinculin (from Laine et al., 1997) 



Full-length vinculin has a rigid 90 kDa head region and a 30 kDa tail region defined by a 
proteolytic cleavage site. As shown, the tail region is folded to indicate the latent binding 
site for F-actin as proposed by Johnson and Craig (1995). Below is a linearized 
representation of human vinculin indicating the reported binding regions and the proteolytic 
cleavage site that generates the p90 head and p30 tail fragments. The head-fragment binds 
monoclonal anti-vinculin 11-5 antibody (Kilic & Ball, 1991), talin (Price et al., 1989), and 

a-actinin (Wachsstock et al. 1987). The location of the IcsA binding site on vinculin's head 
fragment has not been determined. The tail fragment contains the sites for binding F-actin 
(Johnson and Craig, 1995) and paxillin (Turner et al. 1990). Human vinculin also has an 
ABM-1 sequence (residues 840-849) named Vinc-1 located at the carboxy-terminus of the 
p90 fragment generated from vinculin by limited digestion with thermolysin, and included 
for comparison is the second ABM-1 repeat of Listeria ActA (residues 269-278). Also 
shown are the residues (including the conservative substitution of D for E) shared by the 
two sequences. The p30 tail contains two other oligoproline sequences (Vinc-2 and Vinc-3 
that fail to fulfill the consensus features of ABM-1 homology sequences. 



54 

Results 

Anti-ActA-Peptide Immunofluorescence Microscopy Localizes a Cross-Reactive Protein at 
the Back of Moving Bacteria 

VASP was recently shown to concentrate on the surface of intracellular Shigella 
(Chakraborty et al., 1995). To identify the host cell ActA-like protein that binds VASP, we 
used an antibody, designated FS-1, raised against the ABM-1 FEFPPPPTDE sequence of 
Listeria ActA (Southwick and Purich, 1994b). This rabbit polyclonal antibody specifically 
reacted with the ActA surface protein in Listeria cell wall extracts and bound to the surface 
of both intra- and extra-cellular Listeria, as demonstrated by immunofluorescence 
microscopy. The same antibody did not cross-react with Shigella or with cell-free extracts 
of Shigella grown in bacterial culture (data not shown). Immunofluorescence micrographs 
of Shigella-inf&ctQd PtK2 cells demonstrated the presence of a cross-reactive protein or 
proteins on intracellular bacteria (See Figures 3.1A-C). Moving bacteria with F-actin 
rocket tails were identified by TRTTC-phalloidin staining (Figure 3.1 A, rhodamine channel) 
and by phase contrast microscopy (Figure 3. IB). The same bacteria were also stained with 
the anti-ActA peptide antibody FS-1 (Figure 3.1C, fluorescein channel). Of 56 bacteria 
with actin filament rocket tails, 54 demonstrated focal staining with the FS-1 anti-ActA 
antibody. As previously reported (Goldberg et al. 1993, Suzuki et al. 1995, D'Hauteville 
et al. 1996), the IcsA surface protein is shed from the bacterial surface into the actin tails, 
and the cup-like staining pattern observed in Figure 3.1C reflects the same distribution that 
is observed with IcsA (i.e., the greatest concentration was found immediately behind the 
motile bacteria). These observations suggested that the intracellular Shigella bacterium does 
attract a mammalian ActA homologue to its surface, as suggested by our earlier inhibition 
experiments, chapter 2, with ABM-1 and ABM-2 oligoproline peptides (Zeile et al. 1996). 

Dr. F.S. Southwick also found that microinjection of the FS-1 antibody (at 
intracellular concentrations as low as 40 nM) into Listeria-infccted PtK2 cells rapidly halted 
bacterial locomotion, suggesting that the antibody binds to an ActA region that is critical for 



55 

actin-based Listeria motility (unpublished findings). The same intracellular concentration 
of the anti-ActA antibody also rapidly blocked Shigella motility in PtK2 cells (Figure 
3. ID). Microinjection of the same concentration of FS-1 antibody preincubated with a 2 x 
molar concentration of the Listeria ActA ABM-1 peptide (FEFPPPPTDE) failed to block 
bacterial intracellular movement (data not shown), thereby excluding nonspecific inhibition 
by IgG. 

TcsA Affinity Chromatography and Solid Phase Binding Assays Fail to Isolate or Identify 
an ActA Mammalian Homologue 

It was initially hypothesized that the ActA mammalian homologue would bind the 
surface protein IcsA of Shigella with high enough affinity that the application of affinity 
chromatography and/or solid phase assays would allow for its rapid isolation and 
identification. With this in mind, a recombinant IcsA histidine tagged fusion protein using 
an E. coli pET expression system was purified for use in both assays, see materials and 
methods. In the solid phase assay, IcsA was metabolically radiolabeled and used to probe 
total proteins from subcellular PtK2 fractions immobilized on PVDF membranes. In 
repeated assays, we were unable to verify a specific protein interaction with the labeled 
IcsA probe. In addition, when subcellular fractions of mammalian cells were applied to 
the prepared IcsA affinity column, no proteins were specifically eluted. Although at first 
frustrating, it was later appreciated that the binding partner of IcsA or the ActA mammalian 
homologue, must go through a processing step before it is competent to bind with high 
affinity and that even after processing it is present in very low concentrations in the 
cytoplasm of infected cells, see below. 
Identification of a Cleaved Form of Vinculin. p90 as the ActA Mamm alian Homologue 

The ActA-binding protein VASP is abundant in platelets (Reinhard et al., 1992), 
and we expected that one or more ActA-homologue(s) might be present in higher 
abundance in platelets relative to PtK2 cells. Concurrent with my work, Dr. Ron Laine 
used the FS-1 antibody to identify ActA-homologue(s) in platelet membrane extracts that 



56 

were subjected to EEF and subsequent SDS-PAGE. After electrotransfer of proteins from 
the two-dimensional gel, an immunoblot revealed the presence of a major cross-reactive 90 
kDa polypeptide with an approximate isoelectric point of 6.0-6.3 (Figures 3. IE & F). A 
second, less-abundant 53 kDa protein (with a slightly more acidic isoelectric point) reacted 
more weakly with the FS-1 antibody. 

The 90 kDa polypeptide (designated hereafter as p90) recognized by FS-1 anti-ActA 
peptide antibody was collected by excising the Ponseau S-stained protein from the two- 
dimensional electrophoresis blot by Dr. Laine. Upon his finding that the N-terminus was 
blocked, the p90 species was treated treated with Lys-C protease. Subsequent gas-phase 
microsequencing yielded five different peptides (281-GXLRDPSAXPGDAG; 315- 
ERREILGTXK; 417-IAELXDDPK; 607-LLAVAATAPPTDAPNREEVF; 815- 
SFLDSGYRILGA-826), and all corresponded exactly to the numbered positions within 
human vinculin (Weller et al. 1990). 

As a further test of the FS-1 antibody's specificity for the 90 kDa vinculin 
fragment, Western blots of thermolysin-cleaved and intact vinculin were performed by Dr. 
Laine. Johnson and Craig (1994; 1995) reported that vinculin's tail region is folded over 
and many binding sites for known actin -regulatory proteins, as well as internal epitopes, 
are masked by interactions between the head and tail protein (See Figure 3.2). Dr. Laine 
found anti-ActA-peptide antibody only weakly cross-reacted with full-length vincuhn, yet 
reacted strongly with the 90 kDa head-fragment. The C-terminal region of the p90 vinculin 
domain also contains an ABM-1 oligoproline sequence (PDFPPPPPDL, designated Vinc- 
1) that bears sequence homology to the Act-A peptide used to generate the FS-1 antibody. 
Two other oligoproline sequences (named Vinc-2 and Vinc-3) are also found in vinculin 
p30 tail (See Figure 3.2). These proline-rich sequences are distinctly different from Vinc-1, 
because they have intervening basic amino acids that were not recognized by the FS-1 



Figure 3.3. Immunofluorescence microscopy of Shigella-infected PtK2 cells using anti- 
vinculin antibody. 



(A) Fluorescence image of Shigella-infected PtK2 cells using bodipy-phalloidin to label 
polymerized actin. The thin white lines demarcate the junction between the bacterium and 
the actin rocket tail. The asterisk identifies a bacterium that has a small focal cluster of F- 
actin. Bar = 10 |J,m. 

(B) Phase-contrast image of the same field as shown in Panel A. 

(C) Indirect immunofluorescence micrograph of the same cells using the anti-vinculin 1 1-5 
antibody (monoclonal antibody directed against the head-fragment). Note that this anti- 
vinculin antibody localizes to the F-actin tails and to the focal F-actin cluster at one end of 
the bacterium identified by the asterisk. 

(D) An additional fluorescence image of Shigella-Mected PtK2 cells using bodipy- 
phalloidin. 

(E) Same cell visualized by indirect immunofluorescence using the vinll-5 antibody. 



58 






59 

antibody, even on Western blots. In this work, he concluded that the full-length vinculin 
refolds to mask its ABM-1 site, after transfer to the PVDF membrane and exposure to 
physiologic buffer. 
Vinculin's Head Domain Localizes to the Surface of Intracellular Shigella 

To learn whether the vinculin head-fragment serves as a mammalian ActA- 
homologue involved in Shigella motility, bacteria moving in PtK2 cells were studied by 
immunofluorescence microscopy using a monoclonal antibody (Vin 1 1 -5) whose epitope 
has been mapped to the 90 kDa head of vinculin (Figure 3.2). Moving bacteria were 
identified by TRITC-phalloidin staining (rhodamine channel), and the vinculin head- 
fragment was localized (Figure 3.3) by using Vin 1 1-5 antibody staining (fluorescein 
channel). As previously observed with the FS-1 antibody, nearly all moving bacteria 
(identified by the presence of actin filament rocket tails) also were stained with this anti- 
vinculin antibody. Combined with the results with the FS-1 antibody, this suggests that 
moving bacteria have usurped a form of vinculin containing the unmasked Vine- 1 
oligoproline sequence. We had shown that the monoclonal Vin 11-5 antibody did not 
cross-react with any Shigella proteins and it specifically cross-reacted with intact vinculin 
and the p90 head-region on Western blot analysis of PtK2 cell extracts (see Figure 3.5). 

Microinjection experiments were designed on the basis of our past experiments 
(chapter 2), to look at the potential functional significance of the ABM-1 sequence in 
vinculin p90 in Shigella motility. We examined by microinjection of Shigella infected PtK2 
cells, the inhibitory properties of the peptide,Vinc-l, a synthetic peptide based on the 
vinculin ABM-1 oligoproline sequence PDFPPPPPDL. As noted above, this sequence is 
located at the carboxyl-terminus of the p90 head-fragment and shows homology to the 
oligoproline sequences of Listeria ActA (Figure 3.2). In chapter 2 (Zeile et al., 1996) it 
was observed that microinjection of the ActA ABM-1 peptide FEFPPPPTDE arrested 
intracellular Shigella motility at submicromolar concentrations. The Vinc-1 peptide also 
inhibited intracellular bacterial movement in PtK2 cells. Complete inhibition was observed 



60 

at an estimated intracellular concentration of 800 nM Vinc-1 peptide (mean velocity pre- 
injection 0.1 1 ± 0.05 |im/sec, mean and standard deviation, n = 44 versus 0.00 |j.m/sec, n 
= 44 post-injection) (Figure 3.4). Further examination revealed that a ten times higher 
concentration of Vinc-1 peptide was required (inset to Figure 3.4) to produce the same level 
of inhibition observed with the ActA ABM-1 peptide (i.e., half-maximal inhibition was 
observed at 0.5 pM for Vinc-1 versus 0.05 [iU for the ActA ABM-1 peptide). 
Introduction of poly-L-proline (intracellular concentration, \pM) failed to inhibit motility, 
thereby excluding any nonspecific inhibitory effect as in chapter 2 (Zeile et al., 1996). 




to 180 

Time, sec 



270 



Figure 3.4. Effect of Vinc-1 peptide on Shigella speed. 



Representative experiment showing Shigella flexneri speed measurements before and after 
introduction of the Vinc-1 peptide. Arrow indicates time of microinjection of 800 nM Vinc- 
1 peptide, calculated as the intracellular concentration. This experiment is representative of 
multiple determinations (mean pre-injection velocities: 0.1 1 + 0.01, SEM, n=44 vs. post- 
injection: 0.00 ± 0.01 n=44 , p<0.0001). Insert demonstrates the concentration dependence 
of Vinc-1 peptide inhibition. The relative values V(t)A^(0) were determined as described in 
Figure legend 3. ID. Bars represent the standard error of the means (SEM) for 24-44 
observations. 



61 



The Vinculin Head-Fragment is Generated After Shigella Infection 

Although! present in outdated platelets, the p90 vinculin head-fragment was not 
detected in extracts of uninfected PtK2 cells by western blotting. To study how a Shigella 
infection may change the distribution or availability of p90, a time-course of p90 generation 
upon Shigella infection was examined (Figure 3.5 B). The vinculin p90 polypeptide was 
formed and persisted throughout the period over which Shigella axe. typically observed to 
move within the cytoplasm of PtK2 cells (i.e., 1-3 h after the initiation of infection). 
Densitometry scans of the autoradiograms revealed that the p90 proteolytic fragment 
represented 5.6-7.7% of the total vinculin in Shigella-infected PtK2 cells. To exclude the 
possibility that proteolysis was being stimulated by extracellular bacteria, PtK2 cells were 
exposed to a similar number of E. coli and then incubated for 3h. While closely related to 
Shigella flexneri, E. coli lacks the 220 Kb virulence plasmid that permits Shigella to enter 
host cells, and therefore remains extracellular. Infection with E. coli failed to generate the 
p90 fragment in PtK2 cells (Figure 3.5B). Western blots of uninfected and infected cell 

extracts, using an anti-a-actinin antibody, indicated that there were no differences in a- 

actinin proteolysis in uninfected versus infected cells (data not shown). These observations 
indicate that the generation of vinculin p90 did not simply arise from generalized 
proteolysis in infected PtK2 cells. 

Based on Western analysis of Shigella-'miecitd PtK2 cells, it was hypothesized that 
cells containing slowly moving bacteria may be suboptimal with respect to the intracellular 
concentrations of the vinculin p90. Dr. F.S. Southwick tested this prediction by 
microinjecting purified platelet p90 (needle concentration = 1 .2 \xM, estimated intracellular 
concentration = 0.12 |iM) into Shigella infected PtK2 cells. Within 30 s after micro- 
injection, all moving bacteria began to increase their rates and within 60 s, they reached 



62 

velocities that were greater than three times their pre-injection rates . This was the first 
observation of stimulated Shigella locomotion by the addition of a fragment of vinculin. 



Shigella Infection 

(-) (+) (-) (+) 




120 
p90 



Protein Anti-Vinculin 
Stain Western Blot 



B 



Time-course 

t = 1h 2h 3h E.coli 




Anti-Vinculin Western Blot 



Figure 3.5. Shigella infection induces the production of the 90 kDa vinculin head- 
fragment (Part A, from Laine et al., 1997). 



(A) Western blots of PtK2 extracts from uninfected and infected cells using anti- 
vinculin clone 11-5. Left lanes: Coomassie blue stained samples, Right lanes: ECL 
developed Western blots. A 120 kDa cross-reactive polypeptide (full length vinculin) is 
evident in both extracts. However the 90 kDa head-fragment is detected only in the infected 
cell extract. Cells were infected for 3 hours prior to generation of the extract, (from Laine et 
al., 1997) 



(B) Time course of p90 formation after Shigella infection. Note the appearance of 
the p90 band within 1 h of infection. Far right lane shows extract from PtK2 cells infected 
for 3h with a similar number of E. coli. 



63 
Discussion 

A comparison of the actin-based molecular motors of Shigella and Listeria has 
demonstrated marked similarities (Chapter 2); yet it is known that the surface protein, IcsA 
of Shigella, required for motility, has no homology to the ActA surface protein of Listeria. 
Transposon mutation studies identified IcsA as a necessary component for actin assembly 
(Bemardini et al., 1989), and expression of only IcsA on the surface of E. coli stimulates 
actin-based motility in Xenopus oocyte extracts (Goldberg and Theriot, 1995). However, 
IcsA contains none of the ABM-1 VASP-binding sites. Charkraborty et al. (1995) 
demonstrated that Shigella attracts VASP to its surface, and we have confirmed binding of 
VASP to moving bacteria by using anti-hVASP antibody (R. O. Laine, W. Zeile, F. 
South wick, and D. L. Purich, unpublished observations). VASP also has three GPPPPP 
(or ABM-2) sequences for profilin binding, and microinjection of the synthetic VASP 
analogue peptide blocks Shigella actin-based motility (Chapter 2)(Zeile et al. 1996). Kang 
et al. (1997) confirmed that the VASP analogue peptide binds to profiUn using fluorescence 
spectroscopy. These findings indicate that VASP and profilin are key components in 
Shigella 's actin-based motor. 

We have proposed that the IcsA protein must bind an ActA-like adapter protein 
containing one or more ABM-1 sequences to bind VASP. Vinculin is known to bind on 
the surface of intracellularly motile Shigella (Kadurugamuwa et al., 1991; Suzuki et al., 
1996), and IcsA is capable of binding the head-fragment of vinculin (Suzuki et al., 1996). 
Our experiments indicate a role for the p90 head fragment as an ActA homologue that 
attracts VASP. Vinculin p90 fulfills the following essential features of an ActA 
homologue: (a) an ability to bind to IcsA on the surface of Shigella (see Chapter 4), (b) an 
ActA-like ABM-sequence PDFPPPPPDL for VASP binding (Brindle et al. 1996), (c) the 
capacity to be generated by vinculin proteolysis in Shigella-M&ci&d cells, and (d) the ability 
to accelerate actin-based motility of Shigella when introduced into infected cells (Laine et 
al., 1997). These properties suggest a mechanism for assembly of an actin-based motor 



64 

(Figure 3.6). First, proteolysis generates the 90 kDa head, allowing vinculin's newly 
exposed VASP-binding sites to link VASP to Shigella's surface. IcsA has recently been 
shown to have a considerably higher affinity for the p90 head-fragment than for intact 
vinculin (Chapter 4)(Suzuki al. 1996) suggesting that vinculin proteolysis may also 
unmask vincuUn's IcsA binding site. Binding of p90 to Shigella's surface could then 
attract VASP to the same location. Brindle et al. (1996) used GST-fusion protein fragments 
of vinculin (residues 836-940) to demonstrate binding to VASP, and this region of vinculin 
contains the ABM-1 sequence. The oligoproline sequences present in the tail region failed 
to bind VASP (Brindle et al., 1996). 

Sechi et al. (1997) has demonstrated that most actin filaments within bacterial rocket 
tails are organized into long, cross-linked arrays. Their findings disagree with those of 
Tilney and Portnoy (1989) and Tilney et al. (1992a, b) who observed much shorter actin 
filaments in Listeria rocket tails. Based on their findings, Sechi et al. (1997) proposed the 
existence of a polymerization zone on the bacterial surface that accelerates filament 
assembly without increasing spontaneous nucleation or the capture of new filaments. Kang 
et al. (1997) have proposed a cluster model for concentrating VASP and profilin into 
narrow zone on the trailing pole of motile bacteria. They suggest that profilin tethered 
within this zone may reach sub-millimolar concentrations. Recently, Perelroizen et al. 
(1996) suggested that profilin promotes assembly by increasing the efficiency of actin 
monomer addition to the barbed ends of growing filaments, and they conclude that this 
process need not involve profilin-catalyzed exchange of actin-bound nucleotide. In Figure 
3.6 is proposed a mechanism for using vinculin p90 as an adapter molecule for 
concentrating VASP and profilin in a similar polymerization zone on the surface of 
Shigella. 

Inhibition by a synthetic Vinc-1 peptide on Shigella intracellular motility also 
supports the conclusion that the vinculin ABM-1 site is important. In Chapter 2 we 
obtained apparent inhibitory constants (i.e. the concentration needed to inhibit motility by 



65 

50%) of 0.05 )xM and 6 |LiM, respectively, for the Listeria ABM-1 peptide (Phe-Glu-Phe- 
Pro-Pro-Pro-Pro-Thr-Asp-Glu), and the ABM-2 peptide (Gly- Pro-Pro-Pro-Pro Pro). In 
the experiments here, we obtained an approximate inhibitory constant of 0.5 )iM for the 
vinculin ABM-1 peptide (Asp-Phe-Pro-Pro-Pro-Pro-Pro-Asp-Leu) (Figure 3.4). This 
indicates that VASP binds to the Vinc-1 ABM-1 peptide about ten times more weakly than 
the corresponding ActA sequence. This may explain why Listeria form rocket tails having a 
greater F-actin content than Shigella rocket tails (see Figure 2.2, Chapter 2). 

We believe that the ABM-1 peptides do not inhibit bacterial motility by binding to 
Src SH3 domains. SH3 domains interact with two classes of oligoproline ligands, 
designated as the PLRl and RLP2 sequences: APPPLPRR and RALPPLP, respectively 
(Feng et al. 1994). Positively charged guanidinium groups on PLRl and RLP2 interact 
with an essential carboxyl group located on SH3 domains, and this charge-neutralization is 
an essential binding interaction. The negatively charged ABM-1 sequences most likely 
interact with one or more cationic side-chains on VASP. 

It is most likely that the vinculin p90 is the adapter protein required for Shigella's 
actin-based motor. It is also possible that other homologous proteins may also participate 
as a component in an actin-based motor. The cytoskeletal protein, zyxin has several ABM- 
1 sequences (Reinhard et al. 1995b, Purich and Southwick, 1997), but zyxin is about 10- 
30 times less abundant than vinculin in nonmuscle cells (Beckerle, 1986). We have shown 
that vinculin p90 is clearly present at the actin tail-bacteria interface of Shigella, but zyxin 
may also play an analogous role as an adapter. Studies are necessary to further clarify the 
role zyxin and other ABM-1 containing proteins in this process. 

Platelet extract experiments have recently identified a complex involving the actin- 
related proteins (or ARPs) as another potential component of the actin motor (Welch et al., 
1997). This complex may be attracted by the high concentrations of profilin (Machevsky et 
al. 1994) concentrated by both VASP and another VASP-like protein Mena (Gertler et 



66 

al.l996) at the back of Listeria. Similarly, Shigella's ability to attract VASP raises the 
possibility that the ARP complex may be a component of Shigella's actin-based motor. 

Identification of the p90 vinculin head-fragment on the surface of intracellular 
Shigella also raises questions about the abundance of this proteolytic fragment in host cells. 
Western blot analysis fails to detect the p90 proteolytic fragment in uninfected PtK2 cells, 
but this head-fragment is known to accumulate in aging platelets (Reid et al., 1993). The 
latter finding probably accounts for the observation that zyxin is the only VASP binding 
protein in fresh platelets (Reinhard, et al. 1995b). Intracellular infection by Shigella appears 
to be required to generate the vinculin p90 fragment in PtK2 cells, and our studies suggest 
proteolysis may serve as one molecular switch that unmasks the vinculin ABM-1 site. The 
appearance of p90 over the same time-frame as Shigella motility as well as the absence of 
generalized proteolysis point to vinculin proteolysis as a key step in the generation of 
Shigella 's actin based motor. 



Figure 3.6. A working model for vinculin proteolysis and the assembly of the Shigella 
actin -based motility complex (from Laine et al., 1997). 



Intact vinculin contains a set of masked cytoskeletal protein binding sites, including the 
ABM-1 sequence PDFPPPPPDL (shown as a black square beneath the folded C-terminal 
region of full-length vinculin). Proteolysis releases the p90 head-fragment with its newly 
unmasked ABM-1 sequence. Shigella's surface protein IcsA binds the vinculin p90 head- 
fragment. The unmasked ABM-1 sequence then binds the tetrameric protein VASP, which 
deploys its 20-24 ABM-2 sequences to concentrate profilin within a polymerization zone 
located near growing actin filaments in the bacterial rocket tail. The overall ABM complex 
is part of the motor unit which uses bound profilin to usher ATP-actin subunits onto the 
barbed ends of elongating actin filaments. 



68 




feFiilJ-L,«ngtih 



Masked 
AfiM-l Site 






I' 



tr." 
I 



p30 tail 



VineuWn 
p90 Head 



%1 



ABM-I Site 



^•''Z 



L&'y''t6'!csA 





ABM-1 

Site 



-^■-««£ X P9^ 



bacterial 
cell woU 



Rl 



Profifin 







ASSEySLED MOTOR UNIT 




CHAPTER 4 
PRELIMINARY STUDIES OF HOST CELL VINCULIN AND SHIGELLA ICSA 

BINDING INTERACTIONS. 

Introduction 



An activated form of the host cell protein, vinculin, is the adapter protein or ActA 
mammalian homologue in Shigella actin-based motility. Future experiments are planned to 
demonstrate that an actin motor complex can be assembled in vitro from purified proteins 
and cell free extracts; so indicating, the minimal requirements for bacterial actin-based 
motility. We have hypothesized that the minimal components for a Shigella actin motor 
would be cleaved vinculin p90, VASP, profilin, IcsA, and actin, and given proper 
conditions these vv-ould assemble and form a complex that could be detected biochemically. 
In vitro experiments of Kang et al. (1997) demonstrated that profilin bound the GPPPPP 
sequences of VASP with a K^of 84 uM. This would indicate a relatively weak interaction 
and they speculated that this lower affinity is overcome by the bacteria in the cell by 
concentrating profilin to a calculated concentration of approximately 1 mM at the bacterial 
pole-actin tail interface. In this way, the bacterium maintains "an activated cluster "for 
localizing profilin at the bacterial surface due to the multiplicative effect of tethering 
tetrameric VASP with as many as 16 potential profilin binding sites. Therefore VASP is 
the central molecule in the actin motor of either Shigella or Listeria. Because it is 
comprised of four identical subunits, in addition to supplying multiple binding sites for 
profilin, it may also display cooperativity in ligand binding. An allosteric conformational 
change induced by binding of VASP to its target ABM-1 site/s (in the case of Listeria found 
in the four oligoproline repeats of ActA or in case Shigella found in the one oligoproline 
sequence of vinculin p90) may increase the binding affinity for profilin or profilin-actin 



69 



70 

complexes. We speculate that this binding occurs in a sequential manner by assembly of 
the actin motor in specific steps, or an ordered mechanism of ligand binding. In vitro 
assembly of the minimal essential actin motor or actin-based motility complex (ABM 
complex) requires experimental conditions that are optimized to sustain VASP in its high 
affinity or active state by maintaining relatively high concentrations of profilin and the 
addition of components should reflect the order in which they would associate in the cell. 
We have proposed experiments to test this model, the first have been completed and are 
described here. 

The experiments planned required a form of Shigella IcsA that could be readily 
purified and studied by in vitro techniques. Initially, Glutathione S-Transferase-IcsA 
fusion proteins were designed to be purified and then used in solution phase binding and 
chemical crosslinking assays with vinculin. This would be done to establish the usefulness 
of solution phase bead binding experiments for studying these interactions and determine a 
preliminary or approximate dissociation constant for vinculin binding to IcsA. In 
experiments that would follow, IcsA tagged with GST would be used to co-precipitate actin 
motor complexes from cell free extracts and/or purified components. GST-IcsA fusion 
proteins (GIF proteins) were designed to express full length IcsA and various in-frame 
deletion mutants based on our own preliminary work and a published report by Suzuki et 
al. (1996). In this report, similar GST tagged IcsA proteins were used to demonstrate 
vinculin binding domains necessary for actin-based motility. First we hoped to identify 
more precisely the vinculin binding domain on IcsA with a deletion mutant and, second, 
such a mutant would be of lower molecular weight and, possibly, more readily purified and 
manipulated than the full length IcsA. This minimal vinculin binding domain mutant would 
be used in later experiments for studying the in vitro assembly of the ABM complex. 

Here we have characterized, through co-precipitation studies, binding of vinculin to 
full length IcsA fusion protein and evidence for a specific domain for vinculin binding in 



71 

the N-terminal glycine-rich repeat region of IcsA. We also show that modification of 
vinculin by proteolysis is necessary for productive binding to Shigella IcsA in vitro. 

Materials and Methods 

Cloning of Glutathione S-Transferase Fusion Protein cDNA Expression Constructs 

Four BamHI-EcoRI cDNA fragments were generated by PCR from Shigella 
virulence plasmid, pWRlOO template, coding for either full length IcsA or a in-frame 
deletion mutation fused in-frame to the coding region for glutathione S-transferase. The 
cDNA fragment coding for the full length GST-IcsA fusion or GIF construct (GST-IcsA 
Fusion) used in this study, GIF53-758, was amplified by PCR using forward primer 
GIFR53, 5'- CAA ATA GCT TTT GGA TCC CCT CTT TCG GGT- 3' and reverse 
primer GIRV758, 5'- AAG CTG TGA GAA TTC TCA GCG ACT ACT CAT- 3'. This 
cDNA codes for amino acids 53 to 758, which begins the protein coding sequence at the 
first amino acid downstream of the N-terminal signal sequence and stops at arginine 758 in 
the natural cleavage site, SSRRA (Fukuda et al., 1995), upstream of the transmembrane 
coding sequence. The cDNA fragment coding for the N-terminal glycine-rich containing 
region, construct GIF53-302, was amplified using forward primer GIFR53 and reverse 
primer GIRV302 5'- ATATCC GCT ATT GAA TTC TCA CAT ATT GCT- 3'. The 
cDNA fragment coding for an N-terminal deletion of amino acids 1-301 , construct 
GIF302-758 was amplified using forward primer ICFP3 5'- GGT AGC AAT GGA TCC 
ATT GCT AAT AGC GGA- 3' and reverse primer GIRV758. The deletion mutant 
GIF302-418 cDNA fragment was amplified using forward primer ICFP3 and reverse 
primer GIRV418 5'- TAC ATT GAA TTC TCA TGA CAT ATT TCC AGA-3'. PCR 
fragments were digested with BamHI and EcoRI and ligated into expression vector 
pGEX4T-l (Pharmacia Biotech, Uppsala Sweden). E. coli strain BL21 was transformed 
with expression vector constructs and target protein expressed as described in chapter 3. 



72 

Plasmid DNA was isolated from expression clones for confirmation of correct nucleotide 
sequence by DNA sequencing at the University of Florida DNA Sequencing Core. Protein 
expression was monitored by time course analysis and western blotting using an anti-GST 
antibody (Sigma, St. Louis Mo.) 
Recombinant Protein Purification 

GIF53-758 was purified from inclusion bodies using a denaturation-renaturation 
purification protocol. Briefly, after induction and growth of bacterial expression cultures, 
the harvested bacterial pellet was resuspended in lysis buffer, 50 mM Tris-HCl, pH 7.9, 1 
mM EDTA, 1 mM DTT and cells lysed by adding lysozyme to 100 ug/mL and post lysis, 
sonicating to shear the DNA. The bacterial sonicate was solubilized by adding urea to a 
final concentration of 6 M and incubated for 1 h at room temperature. The cell solution was 
then centrifuged at 100,000 xg for 1 h at 4°C. A step dialysis followed to refold the protein 
as described in chapter 3 to a final buffer of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 
mM EDTA, ImM EGTA, 1 mM DTT. After refolding, the protein was applied to a 
glutathione sepharose 4B affinity column or batch purified using glutathione sepharose 4B 
resin (Pharmacia Biotech, Uppsala Sweden) equilibrated in Tris buffer. The column or 
batch resin was washed extensively and bound GST proteins were eluted by applying 
elution buffer, 20 mM reduced glutathione, 50 mM Tris-HCl, pH 7.5, 20 mM DTT and 
fractions collected. Fractions were analyzed by SDS-PAGE, pooled, dialyzed in Q buffer, 
20 mM Tris, pH 7.5, 20 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and applied 
to a Hi-Trap Q 5 mL column (Pharmacia Biotech, Uppsala Sweden). Proteins were eluted 
with a salt gradient, fractions collected, analyzed, pooled and applied to Superose 12 gel 
filtration column equilibrated in GF buffer, 20 mM Tris, pH 7.5, 20 mM NaCl, 1 mM 
EDTA, 1 mM EGTA, 1 mM DTT. Aliquots were flash frozen in liquid nitrogen and stored 
at -70°C. GIF53-302 was purified by the same procedure, except that the denaturation- 
renaturation step was not used as this protein was highly expressed as soluble protein. For 
the ion exchange chromatography step a Mono Q column (Pharmacia Biotech, Uppsala 



73 

Sweden) was used. Crude preparations of GIF302-418 and GIF302-758 were prepared 
only from batch purification using glutathione sepharose 4B. GST protein was prepared 
from batch purification with glutathione sepharose 4B and then by gel filtration on 
Superose 12. 
In vitro Solution Phase Binding Assay: Bead Binding Assay 

Ligand binding reactions were performed with 0.5 uM and 1 uM vinculin (pi 20, 
p90) and 2.5 uM, 5 uM, and 10 uM GIF proteins or GST protein (control). Reactions 
were in a total volume of 200 uL in binding buffer, 20 mM Hepes-NaOH, 150 mM NaCl, 
1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 0.1% BSA. Protein components were mixed 
and 40 uL of 50% slurry glutathione sepharose 4B beads (equilibrated in binding buffer) 
was added and reactions incubated 1 h at room temp with mixing every 15 min by flicking 
the tube. Bound protein complexes were co-precipitated by centrifugation at 500 xg at 
room temp for 5 min. Beads were washed with binding buffer 1 time, centrifuged as 
previously, and washed two times more with binding buffer without 0.1% BSA. After the 
final wash, the supernatant was carefully removed and the beads resuspended in 6X SDS- 
sample loading buffer, boiled 5 min, and loaded on an SDS-PAGE gel. The separated 
proteins were transferred to PVDF membrane by tank transfer as described in chapter 3 and 
western analysis was performed using anti-vinculin clone 11-5 antibody and protein 
complexes visualized by chemiluminescence. 



74 



Results 



Construction and Expression of GST-IcsA Fusion Proteins 

Analysis of Shigella IcsA(see Figure 4.1) (Goldberg et al., 1993)(Fukuda et al., 
1995)(D'Hauteville et al., 1996) has revealed important structural features; a signal 
sequence (amino acids 1-53), a glycine-rich region with repeat motif GGXGGX (amino 
acids 140-307), a specific protease cleavage site SSRIRA (arginine 758-759) and a 
transmembrane sequence beginning at amino acid 759 for anchoring IcsA into the bacterial 
outer membrane. Suzuki et al. (1996) demonstrate that during infection, IcsA is 



SIGNAL SEQUENCE 
30 CKLSFSPFWGASLLLGGPIAFATPLS 56 




140 
172 
206 
240 
274 



PROTEASE CLEAVAGE SITE 
ARG 758 



GSDLSIINQGMI IGGSGGSGA DHNGDGGE-AVT 

GDNLFIINGEIISGGHGGDSYS DSDGGNGGDAVT 

GVNLPI INGKTISGGNGGNNYG EGDGGNGGDAIT 

GSSLSVINKGTFAGGNGGAAYGYGYDGYGGN AIT 

GDNLSVINNGAILGGNGGHWGD AINGSNMTIANS 



GLYCINE-RICH REPEATS 



171 
205 
239 
273 
307 



1102 



Figure 4.1 Schematic representation of Shigella IcsA (from Goldberg et al., 1993) 



cleaved at arginine 758 which releases a 95 kDa fragment that is shed and incorporated into 
the actin tail and that lack of cleavage at one bacterial pole establishes polarity necessary for 
actin assembly in vivo. Although cleavage does occur, full length IcsA on the surface of 
the bacterium is absolutely required for movement by actin assembly (Goldberg and 



75 

Theriot, 1995). Suzuki et al. (1996) further show by deletion mutations of IcsA, that the 
vinculin binding domain is located between amino acids 53 and 506 and the form of 
vincuhn binding with highest affinity is vinculin p90. 

Based on the studies of Shigella IcsA, we designed E.coli expression vectors 
coding for full length IcsA and in-frame deletion mutants for verification of the binding 
domain on IcsA for vinculin. After isolation of the large virulence plasmid of Shigella for a 
template, we used specific primers to generate IcsA cDNAs by PCR for cloning into the 
pGEX4T-l expression vector. Figure 4. 2 A is a schematic of the GST-IcsA fusion 
proteins designed and used in this study. The construct for full length IcsA (GIF53-758) is 
a GST fusion lacking the 52 amino acid signal sequence and the C-terminal transmembrane 
anchor sequence. GIF53-302 is a deletion mutant spanning the glycine-rich region of 
IcsA. If the glycine-rich domain was the vinculin binding site, then a fusion protein was 
designed which eliminated this region but which retained amino acids 302-758 or fusion, 
GIF302-758. Suzuki et al. (1996) left open the possibility that the vinculin binding domain 
was outside the glycine-rich region but before amino acid 506 and so GIF 302-418, a 
deletion mutant containing 1 1 6 amino acids, was constructed. 

In Figure 4.2 B panels 1 -4 are SDS-PAGE gels of cell lysates from expression of: 
panel 1 - GIF53-758, panel 2- GIF53-302, panel 3- GIF302-418, and panel 4- GIF302- 
758. In each panel, lane 1 is cell lysate from cultures before induction by IPTG, lane 2 is 
lysate after 3 hours of induction, lane 3 is protein bound to glutathione sepharose 4B 
beads, lane 4 is a wash fraction and lane 5 is protein after elution from the glutathione 
sepharose 4B. Only expression of GIF53-302 resulted in protein that was primarily 
partitioned to the soluble fraction. The yield from a liter culture of GIF53-302 was greater 
than 10 mg of protein whereas soluble yields from the other three expression cultures 
yielded less than 250 ug per liter of culture. Protein expression from GIF53-758, GIF302- 
758, and GIF302-418 yielded high levels of protein that was partitioned into inclusion 
bodies. We experienced problems that are common to many GST fusion expressions: 



Figure 4.2. Schematic of GST-IcsA fusion proteins used in this study and expression in 
E. coli. 



A) Diagram of wild type Shigella IcsA and GST-IcsA fusion proteins. Construct 1 : Full 
length construct, IcsA amino acids 53 to 758 fused to GST. Construct 2: in-frame deletion 
mutant comprising glycine-rich repeats of IcsA, amino acids 53 to 302 fused to GST. 
Construct 3: in-frame deletion mutant comprising 116 amino acids of IcsA from 302 to 
418. Construct 4: in-frame deletion mutant lacking glycine-rich repeats of IcsA from 
amino acids 302 to 758. 

B) Coomassie stained gels of GST-IcsA fusion proteins expressed in E.coli. Panel 1 : 
Expression of GIF53-758, Panel 2: Expression of GIF53-302, Panel 3: Expression of 
GIF302-418, Panel 4: Expression of GIF302-758. Lane designations for Panels 1-4 are: 
Lane M, molecular weight marker, Lane 1, bacterial lysate pre-induction with IPTG, Lane 
2, bacterial lysate 3h post-induction with IPTG, Lane 3, Soluble fraction from bacterial 
expression lysates. Lane 4, wash fraction. Lane 5, GST fusion protein eluted from beads 
with 20 mM glutathione. 



77 



A) 



Shigella IcsA WT 

53 Glycine-rich repeats 

NH-i — -^ 



H H H tfl 



758 



3-COOH 



1) GIF53-758 



" " " " 



2) GIF53-302 

'^ " " " " ' 



3) GIF302-418 ^ ^^ '•'^ 

4) GIF302-758 302 

GST 



758 



758 




3) M 1 2 3 4 5 




4) 12 3 4 5 




78 

incomplete translation products and proteolytic degradation resulting in a preparation 
contaminated with truncated species of the target protein. Such a mixture is difficult to 
separate by standard purification techniques as these truncated proteins share electrostatic 
and solution phase properties. In addition, it is known that GST itself can associate to 
form dimers causing difficulties for separating a mixture based on size by gel filtration. 

Despite these problems, a method was established for increasing the yield of 
GIF53-758 so that it could be further purified by ion exchange chromatography and gel 
filtration. Briefly, the expression cultures after 3 h of induction were harvested, lysed, 
and then solubilized in 6 M urea. This procedure denatures and solubilizes all of the 
cellular proteins. The DNA is sheared by sonication and the cell lysates are centrifuged at 
high speed to pellet residual insoluble material and membranes. The supernatant is then 
placed in a step dialysis where the urea concentration is slowly diminished over a period of 
24-36 h. During dialysis, the proteins are renatured and some correct refolding occurs. 
The refolded GST tagged proteins are then trapped on a glutathione sepharose 4B resin 
either as a column or batch configuration. Correctly refolded GST fusions should bind the 
glutathione beads, whereas incorrectly folded and proteins lacking the GST domain should 
not bind. The beads with protein bound are extensively washed and then bound protein 
eluted with 20 mM glutathione. The yield from 1 liter of culture at this stage is 15 mg of a 
mixture of target protein and truncated target protein. The eluted protein fractions then are 
size fractionated by gel filtration. After analysis by SDS-PAGE the fractions are pooled 
with a protein yield of 5 mg per liter starting culture. Pooled fractions are then further 
purified by ion exchange chromatography to give a final yield of 0.7-1 .0 mg of protein per 
liter culture. Figure 4.3A is an SDS-PAGE coomassie stained gel of GIF protein 
purifications. Lane 4 is 15 ug of GIF53-758 after employing the above method. In lanes 
2, 3 and 5 are loaded 15 ug of GIF302-418, GIF53-302, and GST, respectively. GIF53- 
302 and GIF302-418 proved to be difficult proteins to purify from the contaminating 
truncated forms and were subject to degradation by proteolysis. Each major band migrated 



Figure 4.3 Purification of GST-IcsA fusion proteins by ion exchange and gel filtration 
chromatography and verification by western blotting. 



A) Coomassie stained SDS-PAGE gel of fusion proteins. Proteins were loaded at 15 ug in 
the following lanes. Lane 2, GIF302-418, Lane 3, GIF53-302, Lane 4, GIF53-758, Lane 
5, GST. Lane 1 and 6 are molecular weight markers. 

B) Western blot using anti-GST antibody with development by alkaline phosphatase and 
visualization by chromogenic substrate (NBT/BCIP). Lane designations are as in (A) 
above. 



80 



A) 



12 3 4 5 6 



200 ■ 

116- 
97.4- 

66 - 



45 - 

31 - 
21.5- 



-'1 



B) 



12 3 4 5 6 



200- 

116- 
97.4- 

66 - 

, 45 • 

31 • 



^U 



81 

at the correct size for each of the target fusion products; GIF53-758 at 109 kDa, GIF53- 
302 at 52 kDa, and GIF302-418 at 40 kDa. Figure 4.3 B is a immunoblot of a membrane 
from a gel identical to Figure 4.3 A which was developed using an anti-GST antibody with 
development by alkaline phosphatase and visualization by chromogenic reagents, Nitro 
Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Sequence analysis of the 
cDNA construct, the correct molecular weight of the target protein on SDS-PAGE, and 
western analysis with anti-GST antibody confirm expression and purification of the correct 
GST-IcsA fusion protein. 
Solution Phase Binding Assay by Co-Preciptiation with Glutathione Sepharose Beads 

In vitro binding of vinculin to IcsA was first studied by binding full length vinculin 
pl20 and vinculin p90 to GST fusion proteins bound to glutathione sepharose 4B beads. 
Figure 4.4 A is a schematic diagram of this assay. After binding of proteins and washing 
to remove non-specific bound proteins, the complexes bound to the beads were solubilized 
in SDS-sample buffer, boiled and loaded on a SDS-PAGE gel for transfer to a membrane. 
The proteins were analyzed by western blotting. The results of three binding experiments 
are represented in Figure 4.4 B in which complex formation was visualized by using an 
anti-vinculin antibody. Figure 4.4B is a western blot where we show that vinculin p90 
binds specifically to the full length IcsA, GIF53-758. Lane 1 is a control binding reaction 
in which 5 uM GST was incubated with 0.5 uM vinculin p90. Lane 2 is a binding reaction 
with 5 uM GIF53-758 incubated with 0.5 uM vinculin p90. From these results, we 
conclude, that in this assay, full length IcsA as a GST fusion protein specifically binds 
vinculin p90. 

Binding of Vinculin to Shigella IcsA Requires the Glvcine-Rich Regi on of IcsA and 
Cleavage of Vinculin to the p90 Form 

We next investigated an IcsA domain for vinculin binding. To accomplish this we 
used the same bead binding assay as previously described, but this time, in addition to full 
length IcsA, the deletion mutants, GIF53-302 and GIF302-418 were tested. We believe 



setpf^SKirT- ■ ■ -■^•vtssss^^vr^y^ 



Figure 4.4 Vinculin p90 binds to full length IcsA and to glycine-rich domain of IcsA. 



A) Schematic of GST bead binding assay. 

B) Vinculin p90, 0.5 uM, purified from human platelets was incubated with 5 uM of 
GIF53-758 and precipitated with glutathione-sepharose. Beads were washed and analyzed 
by SDS-PAGE and immunoblotting using a monoclonal antibody specific for the 90 kDa 
head of vinculin (1 1-5). Lane 1, Binding reaction, GST and vinculin p90, Lane 2, 
Binding reaction, GIF53-758 and vinculin p90. 

C) Binding reactions and visuaUzation as in (B). Vinculin pl20 or p90, 1 uM, was 
incubated with 10 uM of GST fusion proteins. Lane 1, Binding reaction, GST and 
vinculin pl20. Lane 2, Binding reaction, GIF53-758 and vinculin pl20, Lane 3, Binding 
reaction, GIF53-302 and vinculin pl20, Lane 4, Binding reaction, GIF302-418 and 
vinculin pi 20, Lane 5, Binding reaction, GST and vinculin pi 20, Lane 6, 50 ng purified 
p90. Lane 7, Binding reaction. Binding reaction, GIF53-758 and vinculin p90. Lane 8, 
Binding reaction, GIF53-302 and vinculin p90. Lane 9, Binding reaction, GIF302-418 
and vinculin p90. 



83 



A) 



Solution phase binding by GST 
bead binding assay 



Glutathione 
sepharpse 




GST Fusion protein 



B) 



116- 

97.4- 

66 - 



45 









1 2 



C) 



V^ <V^ / 

^ ^ 4" .^ J^ 






(T # # # cy* 4» (^ cf ^ 

123456789 



116- 
97.4- 

66 - 
45 - 



L 



84 

that the glycine-rich region containing the GGXGGX motif, which is repeated seven times 
in the IcsA molecule (see Figure 4.1), may be a region in which multiple vinculin 
molecules could bind, mimicking the four repeats of Listeria ActA. For Shigella motility, 
this configuration may be necessary because vinculin p90 has only one ABM-1 site per 
activated molecule. This multiplicity of binding sites may allow Shigella through the IcsA 
molecule, vinculin p90, and VASP, a way of increasing the effective concentration of 
profilin and profilin-actin complexes at its bacterial actin-tail interface. The clustering of 
profilin would occur in the same manner as it is clustered on the trailing pole of Listeria. 
We therefore tested GIF53-302, an IcsA glycine-rich domain mutant, for its ability to bind 
vinculin p90 or vinculin pi 20. An additional mutant, GIF302-41 8 was also generated to 
the sequence 1 16 amino acids upstream of the glycine-rich repeats. This was done to 
address the findings of Suzuki et al. (1996), that found vinculin binding in the region of 
amino acids 53 to 418 of IcsA, using similar assays. 

Figure 4.4 C is a western blot in which anti-vinculin 1 1-5 antibody was used to 
visualize vinculin pi 20 and p90 binding during complex formation with the GST fusion 
proteins. In these binding reactions 1 uM vinculin either as p90 or pi 20 was incubated 
with 1 uM of the GST fusion proteins. Comparison of Lane 3 in which the binding 
reaction contained vinculin pl20, and GIF53-302, to Lane 2 containing vinculin pl20 and 
GIF53-758, and Lane 4 containing vinculin pl20 and GIF302-418, demonstrates that the 
glycine-rich mutant binds with highest affinity to vinculin pi 20 and that the deletion 
mutant, GIF302-418 does not bind vinculin pi 20. Lanes 1 and 5 are control lanes 
containing GST and vinculin pl20 binding reactions. Lane 7 is the binding reaction of full 
length IcsA fusion with vinculin p90, Lane 8 is the binding reaction of the glycine-rich 
mutant, GIF53-302 and vinculin p90, and Lane 9 is the binding reaction of vinculin p90 
and GIF302-418. These results indicate that vinculin p90 binds with highest affinity to the 
glycine-rich mutant, G1F53-302, and full length IcsA, GIF53-758, but does not bind to 
GIF302-418. Overall, the results are in agreement with Suzuki et al. (1996), in which they 



85 

also found the most likely binding domain on IcsA for vinculin to be the region of glycine- 
rich repeats. These experiments indicate that the presence of the vinculin 30 kDa tail on 
uncleaved vinculin is sufficient to block binding of full length IcsA and that either by 
removing the 30 kDa tail to form p90 or truncating of IcsA allows for a higher affinity 
binding interaction. These results are consistent with our previous findings in which we 
show that vinculin pi 20 must be cleaved to form the active p90 form that binds IcsA in 
vivo. 

Discussion 

The pathogenic bacteria, Listeria monocytogenes and Shigella flexneri are able to 
multiply and spread in host cells due to their ability to subvert the host cells' cytoskeleton 
for their own actin-based motility. We have demonstrated that the mechanism that is 
employed by these bacteria is essentially the same (Chapter 2). These organisms during 
their intracellular life cycle exploit similar host cell proteins to assemble their actin motors. 
In both cases, profilin and profihn-actin are required for this process. In both cases 
tetrameric VASP is required for this process and in both cases a specific bacterial surface 
protein is required for this process. An important finding is, that despite a conserved 
mechanism, there is an additional host cell protein that only Shigella requires. This may 
further demonstrate the conserved nature of directed actin polymerization in which the basic 
actin motor is conserved, but with minor variations to adapt it to different cell types or 
organisms. It is known that other organisms such as Rickettsia, enteropathogenic E. coli 
(EPEC), and the vaccinia virus also direct actin assembly for intracellular 
movement(Southwick and Purich, 1996)(Cudmore et al, 1995), and although their 
mechanisms for exploiting host cells is not completely understood, it is likely that they use 
a similar mechanism to that o^ Listeria and Shigella (Finlay and Cossart, 1997). 

In this last series of experiments we have presented additional evidence that vinculin 
within the host cell must be activated by proteolytic cleavage for productive binding to 



86 

Shigella IcsA during infection. This was demonstrated by glutathione bead binding assays. 
In these experiments it was shown that the highest affinity binding interaction was between 
IcsA or a specific domain of IcsA and the activated form of vinculin, vinculin p90. These 
assays alone are not sufficient proof that such an interaction takes place upon Shigella entry 
into a host cell; but they support our previous immunofluorescence data, microinjection 
data, cell culture infection data, and immunological data. 

We have also presented evidence that the domain on IcsA to which vinculin p90 
binds is the glycine-rich region of the repeated motif GGXGGX. As alluded to earlier, the 
significance of binding in this region is that multiple vinculin p90 molecules may need to be 
bound to IcsA so that VASP can be recruited and bound in its active conformation. 
Sustaining the active conformation of VASP may require that VASP be bound at more than 
one ABM-1 site. Quantitative analysis of the binding experiments may reveal multiple 
molecules of vinculin bound in this region of IcsA which would argue for intramolecular 
binding site interactions. It is also possible that vinculin binds IcsA in a 1 : 1 stoichiometry, 
yet VASP is activated due to intermolecular interactions of vinculin residing on adjacent 
IcsA molecules. In either case, careful analysis may distinguish between these 
possibilities. Further experiments are necessary to establish the role of ligand binding in 
the transition of VASP between inactive to active states during assembly of the actin motor. 
If this could be demonstrated experimentally, VASP could be added to the growing hst of 
proteins such as, hemoglobin and aspartate transcarbamoylase, that are regulated by 
allosterism. 

Beyond characterizing a binding domain on IcsA for vinculin p90, we have 
endeavored to design a system in which in vitro assembly of an actin-based motility (ABM) 
complex can be demonstrated. See Chapter 5 and Figure 5.1 for a further discussion of the 
ABM complex and our model for actin-based motility. We now have in place tagged 
molecules which can be readily manipulated and when supplied with the correct 
components and with the proper conditions should form an ABM complex. The following 



87 

experiments are proposed. To assay for complex formation, native VASP will be co- 
precipitated from platelet extracts using GST-IcsA fusion protein, GIF53-758, bound to 
glutathione sepharose beads in the presence of exogenous vinculin p90 and profilin. As 
the source of native VASP, freshly outdated platelets are lysed and extracted with high salt 
to enrich for membrane bound proteins. The amount of VASP present in 1 unit of platelets 
is approximately 100 ug which is sufficient for the proposed experiments. To remove 
endogenous vinculin, the extract is mixed with a anion exchange resin and then filtered. It 
would be important at this point to remove endogenous zyxin, but with a basic pi very 
close to VASP it would not be possible to remove zyxin from the extract without removing 
VASP as well. Although zyxin contains multiple ABM-1 sites it is about 10-30 times less 
abundant than vinculin (Beckerle, 1986) in non-muscle cells and therefore may not be a 
confounding factor is these assays. Before use the prepared platelet extract is centrifuged at 
high speed to pellet F-actin. This step is required to eliminate false-positive complexes that 
result in pelleting F-actin and its associated actin-binding proteins. The binding reactions 
are then assembled with vinculin p90 and GIF53-758 to a final concentration of 5-10 uM in 
a reaction volume of 200 uL. After a brief incubation, glutathione sepharose beads are 
added, incubation is continued with intermittent mixing, and then the complexes are 
pelleted by centrifugation. The pelleted beads with bound complex are washed and after 
the final wash resuspended in SDS-sample buffer and loaded on a gel. The separated 
proteins are transferred to a membrane and visualized by western blotting and 
chemiluminescence. 

Assuming a relatively conservative K^ of 100 uM a complex of 0.01 ug/uL to 0.1 
ug/uL could be formed which would be within the detection limits of western blotting and 
visualization by enhanced chemiluminescence. Reaction and wash conditions would be 
varied with respect to the exogenous profilin concentration. A series of replicates would be 
done for each reaction and wash condition. All components kept at a constant 
concentration would be; platelet extract, exogenous vinculin, and GST-IcsA fusion protein. 



while the exogenous profiUn concentration would be varied. A first experimental set would 
have the components without exogenous profilin in the reaction and washes. A second set 
would have exogenous profilin added to the reaction but not the washes. The third set 
would have exogenous profilin in the reaction and the washes. We hypothesize that 
profilin is required in both the reaction and the washes to maintain VASP in its active and 
therefore high affinity state throughout the manipulations. If this is true, at lower 
concentrations of profilin, less complex should form. These conditions would exist either 
in the experimental sets which have low profilin concentration in the reaction and washes or 
in the experimental sets in which profilin is omitted from the wash steps. 

Misleading results in these assays may arise from a number of causes including, co- 
precipitating actin and actin binding proteins. As a control, the specific ABM-1 and ABM- 
2 synthetic peptides can be used as competitive inhibitors in the binding reactions. In this 
case, positive complex formation can be tested by titration with these synthetic peptides to 
an appropriate concentration that disrupts the interaction. Our microinjection experiments 
in which we inhibited bacterial motility with these peptides can be used as a guide to 
determine the inhibitory concentration of synthetic peptide that should be used. For an 
additional control, we have available a mutant profilin that is impaired for binding poly-L- 
proline. This profilin should not bind VASP and therefore in the binding reactions should 
not form an ABM complex. This would be added proof that the complex formed in the 
presence of wild type profilin was specific. 

If successful, these experiments would support our model for a minimal actin motor 
consisting of: actin, profilin, VASP, and an ABM-1 site containing protein. In the next 
chapter we will discuss this further and explore other experimental approaches to 
investigating actin-based motility and its regulation. 



CHAPTER 5 
SUMMARY AND FUTURE DIRECTIONS 

After escape from the phagolysosome, Shigella begins assembly of an actin motor 
from host cell components. It is known that IpaA, a secreted protein of the Ipa family of 
invasin proteins of Shigella, interacts with host cell vinculin to mediate efficient 
internalization of the bacterium and that vinculin is redistributed from the cytoplasmic pool 
to sites of bacterial entry during early stages of the infection (Tran Van Nhieu et al., 1997). 
Tran Van Nhieu et al. (1997) also found that vinculin was localized around the bacteria 
very early in the infection well before actin assembly. Processing of vinculin may be 
required at this stage, for modifying vinculin to a form with high affinity for IcsA. Shigella 
has to compete with talin, an anchoring protein of focal adhesions, for vinculin and only by 
generating a form of vinculin with more favorable binding interactions or lower K^ for 
IcsA, could it assemble its actin motor. Regulation of vinculin unfolding by 
phosphorylation (Schwienbacher et al., 1996) or by phosphoinositides, namely 
phosphatidylinositol (4,5)-bisphosphate [PtdIns(4.5)P2] (Gilmore and Burridge, 1996) to 
unmask F-actin and paxillin binding sites has been proposed for actin remodeling in a 
normal cell. We now believe that Shigella either directly or indirectly initiate a proteolytic 
processing pathway for vinculin. Shigella during infection may exploit programmed cell 
death or apoptosis during which the release of proteolyzed forms of cellular proteins are 
made available. Bacterial induced apoptosis of macrophages by the invasin, IpaB of 
Shigella, has been demonstrated in vitro (Zychlinsky et al., 1996). IpaB binds to 
interleukin-lB converting enzyme (ICE), a cysteine protease, [now referred to as caspase-1 
(Cohen, 1997)] known to initiate the proteolytic cascade of apoptosis (Martin and Green, 



89 



90 

1995). A number of substrates have been identified for caspases and these are cleaved at 
an aspartate residue within the cleavage motif, DXXDI X. Amino acid sequence analysis 
failed to uncover a similar motif in vinculin, but this does not preclude the possibility that a 
caspase may be involved in cleavage of vinculin. Calpain, another cysteine protease, 
which is an intracellular, calcium dependent protease (Inomata et al., 1996) is activated and 
cleaves cytoskeletal proteins under conditions of elevated intracellular calcium or during 
apoptosis (Martin and Green, 1995) and may also be involved. Cell death can also occur 
upon Shigella infection by the secretion of the ricin-like depurinase or shiga toxin of 
Shigella which blocks protein synthesis (Gyles, C.L.,1993). Induction of a proteolytic 
cascade by apoptosis may be a necessary feature of Shigella actin-based motility. 

We would like to identify the protease that specifically cleaves vinculin during 
Shigella infection. Identification of this protease would add further evidence for the role of 
proteolysis in Shigella actin motor assembly and would define a more general mechanism 
for specific activation of regulators of actin polymerization. The following are two possible 
experimental approaches for isolating and characterizing this specific protease. 
In vitro Expression Cloning to Isolate a Specific Protease 

Recently described is a technique known as in vitro expression cloning 
(IVEC)(King et al., 1997), which uses biochemical functional assays to screen cDNA 
expression libraries to clone genes of proteins involved in specific protein-protein 
interactions. Because many cellular processes involve post-translational modification of 
protein substrates such as by phosphorylation and proteolysis, common functional assays 
can be used to screen an expression library for a specific modification activity. In so doing 
the gene for that specific activity is cloned. Briefly, cloning by this method is as follows. 
A cDNA expression library is subcloned into an expression plasmid vector or a phagemid 
cDNA expression library is constructed or purchased. Plasmid DNA is purified and the 
plasmid library divided into pools of 50-100 clones. The pooled plasmids are expressed in 
an in vitro coupled transcription-translation system. The expressed protein pools are then 



91 

screened for biochemical activity. The screening is repeated until a single cDNA encoding 
the activity is isolated. Among proteins that have been identified this way are; specific 
substrates for caspases (Kothakota et al.,1997), phosphorylation substrates, substrates 
degraded by cell cycle specific proteolysis (King et al., 1997), and bacterial proteases 
(Shere et al., 1997). 

This method may prove useful in screening for the protease that is activated during 
infection by Shigella. In vitro translated protein pools can be screened for the ability to 
cleave vinculin pi 20 to p90. Screening of proteolysis reactions can be done in 96 well 
microtiter plates in which at first screening a recombinant library of 200,000 clones can be 
assayed with 40-20 plates at 50-100 clones per well. After screening, the cleavage can be 
monitored by SDS-PAGE and coomassie staining. Some important assumptions are made 
in using this assay for identifying biochemical activity. It is assumed that the proteins 
translated in this system are correctly folded and they require no additional modification for 
activity. Also, if the proteolytic activity required a multi-subunit protein complex; these 
proteases would not be found in such a screen. Large numbers of false positives may be 
another problem with this method. The structure of vinculin makes it susceptible to 
cleavage by proteases in the region of amino acids 830-890 which spans the oligoproline 
repeats. To address this possibility, positive clones could be screened in conjunction with 
the FS-1 anti-ActA antibody. Only vinculin molecules with an intact ABM-1 site would be 
detected and only those plasmid pools would need to be screened further. Additionally, 
the assay could be scaled up analyze many more recombinant clones by using an ELISA 
based assay, in our case, based on the FS-1 antibody and screening with a microtiter plate 
reader. Another variation of the method would look for proteins that specifically bind 
vinculin by immobilizing vinculin and assaying for expressed proteins that bind with high 
affinity. 



92 
Biopanning a Phage Display Library 

A second approach to isolating proteases involved in vinculin cleavage, or even 
more significantly, other binding partners of cytoskeletal proteins we have determined to be 
important in actin-based motility would use a screen for protein-protein interactions by 
biopanning a phage display library (Smith and Scott, 1993). Using this methodology, 
peptides or proteins are incorporated into the coat protein of infective E.coli fd filamentous 
or T7 phage. The phage with the foreign peptide or protein displayed on its surface is 
introduced to possible ligands that are bound to a solid support. The binding reactions are 
washed to remove non-specifically bound phage particles and then bound phage are eluted. 
The eluted phage from the first screen are amplified and reacted in a second or third screen. 
At this point, phage enriched for the specific binding interaction can then be infected into 
E.coli and the DNA purified by standard methods and then sequenced. The power of the 
technique is that a completely random peptide or polypeptide library can be generated 
making use of all 20 amino acids. This is done by incorporating degenerate oligonucleotide 
inserts into the coat protein genes. Greater than 1 million random hexapeptides have been 
expressed on the pIII protein of the filamentous phage, fd. These were generated from 
synthesized oligonucleotides of the sequence (NNK)g, where N=A, T, C, G and K=T or G 
to make up a library of 3 x 10^ recombinants (Cwirla et al, 1990). It is also possible to 
create a peptide or polypeptide library around a conserved core or motif. In this case, a 
biased oligonucleotide sequence is specified such as in our case may be, (CCN)4, for the 
oligoproline sequence, PPPP, and then the flanking sequences randomized by supplying 
equimolar mixtures of the nucleotides to the synthesis reaction (see below for further 
discussion). 

With vinculin pl20 or p90 immobilized on a solid support, random hexapeptides or 
larger polypeptides could be analyzed for the preferred binding partner. This screening 
may reveal an amino acid sequence found in the active site of a specific protease. 
Expressing a vinculin fragment encompassing only the oligoproline region would make the 



93 

screen more specific for the protease in question. Although biopanning a phage display 
library would be less specific for isolating the vinculin protease, the information obtained 
about the preferred amino acid composition of bound polypeptides would greatly expand 
our knowledge of how protein domains interact with vinculin for productive associations. 
Biopanning a Phage Display Library Based on a Biased Oligonucleotide Sequence 

By changing the bound ligand and generating a phage display library from a biased 
oligonucleotide sequence; biopanning can discover preferred protein-protein interactions 
based on a pre-determdned binding motif. In Chapter 1 , it was discussed that oligoproline 
sequences have unique properties that make these sequences ideally suited for protein- 
protein interactions. As mentioned, poly-proline binding is favored both enthalpically and 
entropically, and flanking amino acids play an important role in defining the specificity of 
the binding interaction. We believe that charged residues are critical for defining this 
specificity as discussed in Chapter 3. There we discussed that SH3 domain binding 
oligoproline peptides containing positive charges do not show effects on bacterial motility 
in our microinjection assays and therefore the negative charges of the ABM-1 sequences 
must be critical for defining the specificity of the interaction required for assembly of the 
actin motor. With VASP, domains of VASP, or profilin bound to a support; mutant ABM- 
Kin the case of immobilized VASP) and ABM-2 (in the case of immobilized profilin) 
peptides built around a conserved PPPP core displayed on fd phage or T7 phage could be 
screened for those sequences with highest binding affinity. Analyzing these sequences 
would clearly reveal the important components, such as charge and/or hydophobicity, of 
the binding interactions of ABM-1 and ABM-2 sequences. 

Another Model System: Vaccinia Virus 

A number of life forms have exploited actin-based motility to gain a competitive 
edge in their ecological niche. As mentioned in earher chapters, Cudmore et al. (1995) 
have described the ability of the orthopoxvirus, vaccinia virus to use actin assembly for 



94 

intercellular spread. Vaccinia virus is a double stranded DNA virus that replicates and is 
assembled entirely in the cytoplasm of host cells. The complete genome of 200,000 base 
pairs has been sequenced with coding for approximately 260 open reading frames (ORP). 
Blasco et al. (1991) recognized that one of these ORF's coded for a homologue of 
mammalian profilin, viral protein A42R. They went on to generate mutant virus in which 
A42R was deleted and demonstrated that this profilin homologue was not necessary for 
actin-based motility by the virus nor was it necessary in cell-to-cell transmission. Other 
ORF's have been analyzed for homologies to cytoskeletal proteins identified in actin-based 
motility of other organisms, but to date none of the viral ORF's code for proteins necessary 
for this activity. Wolffe et al. (1997) and Reckmann et al. (1997) have both performed 
experiments looking at viral proteins thought to be necessary for actin assembly. Viral 
proteins analyzed by these groups, F17R and A34R, proved not to be necessary for viral 
actin-based motility, although in the case of F17R some actin bundling activity was 
observed and A34 R was found to be necessary for efficient viral transmission. 

The membranes surrounding the different virion forms are complex. MaUare 
cytoplasmic virions or intracellular mature virions (IMV) are enveloped in a double 
membrane that is acquired from the intermediate compartment between the endoplasmic 
reticulum and the golgi network of the host cell. IMV s are further surrounded by 
membranes from the golgi cisternae to form the intracellular enveloped virion which 
becomes the infectious form when it fuses with the plasma membrane. Infectious virions 
when associated with the membrane are cell-associated enveloped virions and when shed 
outside the cell are extracellular enveloped virions. However complicated the membrane 
envelope of the mature virus, the outer membranes are host cell derived. It is entirely 
possible that the virus obtains all the proteins necessary for actin-based motility from the 
host cell and may not need to have a specific surface protein, only the ability to generate 
active host cell proteins such the vinculin p90 that we propose is generated upon Shigella 
infection. 



95 

We propose experiments to look at the process of vaccinia actin-based motility from 
a cellular standpoint. We know that VASP is present in the bacterial actin motor and in 
host cell actin assembly. Assuming that VASP is the central molecule in any actin motor 
one would expect to find it at the interface of the actin tail and the motile organism. In 
unpublished results of Higley and Way (1997), they claim to have localized VASP to the 
viral-actin tail interface and therefore we propose to look by immunofluorescence for VASP 
at the vims-actin tail interface in infected cells. Secondly, we would look for vinculin p90 
with the FS-1 antibody and the anti-vinculin clone 1 1-5 antibody. Vaccinia particles with 
surface proteins lacking ABM-1 sites may behave like Shigella and recruit an ActA-like 
molecule in order to bind VASP and this molecule may be vinculin. Another ABM-1 site 
containing protein is zyxin and using the anti-zyxin antibody we would also look for the 
presence of zyxin at the virus-actin tail interface. Although the viral encoded profilin 
homologue was found not to be necessary for actin-based motility by the virus, more than 
likely, the host cell profilin is recruited and therefore, we would look for mammalian 
profilin at the virus-actin tail interface. With these experiments it would be possible to 
include vaccinia virus in the group of life forms that use a mechanism of actin-based 
motility that is now defined by our model. 

The Actin-Based Motility Complex 

The central role in actin dynamics in general and bacterial actin-based motility in 
particular, is played by vasodilator-stimulated phosphoprotein (VASP). VASP first was 
shown to prevent activation of platelets and blood vessel endothelial cell constriction when 
phosphorylated in response to vasoactive agents such as cGMP or cAMP (Reinhard et al., 
1992) and localized to sites of focal contacts and in lamellae of locomoting cells. 
Chakraborty et al. (1995) next demonstrated a direct interaction between Listeria ActA and 
VASP and also showed a colocalization of VASP with intracellular Shigella, but were 
unable to demonstrate a direct interaction of VASP with Shigella. This suggested at the 
time that Shigella required an additional host cell protein for VASP binding. As we have 



96 

seen, this host cell protein is an activated form of vinculin. At the same time, Reinhard et 
al. (1995) revealed that VASP was a substrate for binding profilin at the GPPPPP repeats 
of VASP. This was an important observation because it could explain the findings that 
profilin could be localized to Listeria-actin tail interface in infected cells when profilin could 
not be shown to bind Listeria in vitro (Theriot et al., 1994). Different models of actin 
assembly, including our own, required a mechanism for concentrating profilin or profilin- 
actin complexes at sites of actin polymerization and a mechanism could now be 
hypothesized in which VASP played the key role in recruiting polymerization competent 
ATP-actin monomers to regions of rapid growth of actin filaments by its binding interaction 
with profilin. 

We now envision the following sequence of events for assembly of an actin-based 
motility (ABM) complex (see Figure 5.1). In the case of Listeria (Figure 5.1 top panel), 
the four oligoproline repeats of Listeria ActA bind tetrameric VASP. Membrane bound or 
cytoplasmic VASP is readily recruited by the bacteria due to the high affinity of VASP for 
ABM-1 sequences (Niebuhr et al., 1997). VASP, initially, may be in an inactive state, but 
when bound to an ABM-1 sequence/s may go through a conformational change to an active 
state. In this activated state, VASP readily binds profilin or profilin-actin complexes and 
actin assembly takes place. For assembly of the Shigella actin-based motility complex 
(see Figure 5.1 bottom panel) the surface protein, IcsA of Shigella, recruits the host cell 
protein vinculin after vinculin' s ABM-1 site has been unmasked by proteolysis. The 
glycine-rich repeats of IcsA may bind multiple vinculin molecules. The necessity of 
multiple bound vinculin molecules with unmasked ABM-1 sites may be to mimic the high 
affinity state of the oligoproline repeats of ActA, in the recruitment of VASP, stimulating its 
transition from an inactive to active state. Once active VASP is tethered to Shigella, 
profilin and profilin-actin is concentrated just as with Listeria. Uncapping of actin 
filaments and action of depolymerization proteins, such as ADF/cofilin, may also be 



97 

required for initiation and maintenance of actin polymerization (Carlier and Pantaloni, 
1997) once the ABM complex is established. 

Our future experiments will try to establish that the ABM complexes we have 
described represent the minimal essential components required for actin-based motility. We 
will also attempt to demonstrate cytoskeletal regulation by an allosteric mechanism for 
VASP in its transition from inactive to active states. In addition, we would like to 
investigate the proteolytic cascade that Shigella initiates during infection and try to discover 
the protease or proteases that may be activated. This work will lead to our understanding 
of how the eukaryotic cell is able to remodel its actin cytoskeleton in response to 
intracellular and extracellular signals. 



98 



Assembly of the Listeria ABM Complex 



VASP 




N 



inactive 



OOqPo o 

OoogSoo 



o 

Profllin 



J ActA 




ABM 
Complex 



/ 



Assembly of the Shigella ABM Complex 



VASP 







inactive 




N 



active 



opogoOo 
o°ooOO 

Profllin 



! 



I 



Vinculin p90 





ABM 
Complex 



/ 



IcsA 



Figure 5.1. Schematic of Actin-Based Motility Complexes of Listeria and Shigella actin 
motors (with permission D.L.Purich, 1997) 



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BIOGRAPHICAL SKETCH 

William L. Zeile was born December 24, 1949, in Flint, Michigan, to parents 
Walter L. Zeile and Ruth Virginia Clemetson. His father, the Reverend Walter Zeile, was 
pastor for Trinity Lutheran Church of Davidson, Michigan. At age 1 1 Will and his family 
moved to Basking Ridge, New Jersey, where his father became pastor of Somerset Hills 
Lutheran Church of Basking Ridge, NJ. Will graduated from Ridge High School of 
Basking Ridge, NJ, and went on to college, first at Bucknell University and then, after 
transferring, to the University of Michigan at Ann Arbor where he received his Bachelor of 
Science degree in 1972. He served in the Peace Corps in Swaziland, Africa, from 1977 to 
1980 as a secondary school teacher in a rural school. During this time he met and married 
his wife, Patricia Mary Comstock. Upon returning from the Peace Corps, he and Patty 
managed and operated a small dairy farm in upstate New York. After being blessed with 
four children Kathleen Ruth, James Walter, Rachel Euseba, and Joshua William, Patty and 
Will moved to Florida where Will entered the University of Florida as a graduate student in 
the College of Agriculture, Department of Agronomy. He received his Master of Science in 
April 1994. That same year he entered the Ph.D. program in the College of Medicine, 
Department of Biochemistry and Molecular Biology. 



106 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




•^t..l /v^v:.iC^,X 




.Tederick S. SouthwiclJ, Chairman 
Professor of Biochemistry and Molecular 
Biology 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. ^/^ 




IJaniel L. Purich 
Professor of Biochemistry and Molecular 
Biology 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 



«S^'./^.' 




Gudrun Bennett 

Research Professor of Anatomy and Cell 
Biology 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Brian D. Cain 

Associate Professor of Biochemistry and 
Molecular Biology 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 





Charles M. Allen 
Professor of Biochemistry and Molecular 
Biology 



This dissertation was submitted to the Graduate Faculty of the College of 
Medicine and to the Graduate School and was accepted as partial fulfillment of the 
requirements for the degree of Doctor of Philosophjj.^/ 

December 1997 




DearirGollege of Medicine 

I/' 



Dean, Graduate School